Compositions and methods for inhibiting GYS2 expression

ABSTRACT

This disclosure relates to oligonucleotides, compositions and methods useful for reducing GYS2 expression, particularly in hepatocytes. Disclosed oligonucleotides for the reduction of GYS2 expression may be double-stranded or single-stranded, and may be modified for improved characteristics such as stronger resistance to nucleases and lower immunogenicity. Disclosed oligonucleotides for the reduction of GYS2 expression may also include targeting ligands to target a particular cell or organ, such as the hepatocytes of the liver, and may be used to treat glycogen storage diseases (e.g., GSDIa, GSDIII, GSDIV, GSDVI, and GSDIX) and related conditions.

RELATED APPLICATION

This application claims the benefit under 35 U.S.C. § 119(e) to U.S. Provisional Application No. 62/637,574, filed Mar. 2, 2018, and entitled “COMPOSITIONS AND METHODS FOR INHIBITING GYS2 EXPRESSION,” the entire contents of which are incorporated herein by reference.

FIELD OF THE INVENTION

The present application relates to oligonucleotides and uses thereof, particularly uses relating to the treatment of glycogen storage diseases and associated conditions.

REFERENCE TO THE SEQUENCE LISTING

The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled D0800.70014WO00-SEQ.txt created on Feb. 15, 2019 which is 132 kilobytes in size. The information in electronic format of the sequence listing is incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

Glycogen is a complex sugar used by the body to store glucose. When the body requires more glucose to function, it normally breaks down the stored glycogen for use in cellular processes. Several enzymes participate in the processes that are used to store glucose as glycogen (glycogen synthesis) and break down glycogen to glucose (glycogen breakdown). When one or more of these enzymes are inhibited, it can result in a glycogen storage disease in which a dearth of glycogen storage, a buildup of glycogen in affected cells (e.g., liver and/or muscle cells), or the formation of abnormally structured glycogen may be observed. When a disorder of glycogen storage or breakdown occurs, those affected may suffer from a number of symptoms including, but not limited to: hepatomegaly, increased liver toxicity (e.g., higher levels of AST, ALT, and/or ALP), liver fibrosis, fatty acid deposition in the liver, hepatic hyperplasia, hepatocellular adenoma, and/or hepatocellular carcinoma. A non-limiting set of exemplary glycogen storage diseases may include: GSDI (e.g., GSDIa), GSDIII, GSDIV, GSDVI, and GSDIX.

BRIEF SUMMARY OF THE INVENTION

Aspects of the disclosure relate to oligonucleotides and related methods for treating a glycogen storage disease (e.g., a disease or disorder affecting glycogen breakdown or storage such as GSDIa, GSDIII, GSDIV, GSDVI, or GSDIX) in a subject. In some embodiments, potent RNAi oligonucleotides have been developed for selectively inhibiting GYS2 expression in a subject. In some embodiments, the RNAi oligonucleotides are useful for reducing overall GYS2 activity in hepatocytes, and thereby decreasing or preventing hepatomegaly, liver toxicity (e.g., levels of AST, ALT, and/or ALP), liver fibrosis, fatty acid deposition in the liver, hepatic hyperplasia, hepatocellular adenoma, and/or hepatocellular carcinoma. In some embodiments, key regions of GYS2 mRNA (referred to as hotspots) have been identified herein that are particularly amenable to targeting using such oligonucleotide-based approaches (See, e.g., Example 1).

One aspect of the present disclosure provides oligonucleotides for reducing expression of GYS2. In some embodiments, the oligonucleotides comprise an antisense strand comprising a sequence as set forth in any one of SEQ ID NOs: 193-384, 417-466, 518-568, 575-580, 586-598, or 620-627. In some embodiments, the antisense strand consists of a sequence as set forth in any one of SEQ ID NOs: 193-384, 417-466, 518-568, 575-580, 586-598, or 620-627. In some embodiments, the antisense strand comprises, or consists of, a sequence as set forth in any one of SEQ ID NOs: 417-466, 575-580, 586-598, 620-627. In some embodiments, the oligonucleotides further comprise a sense strand that comprises a sequence as set forth in any one of SEQ ID NOs: 1-192, 385-416, 467-517, 569-574, 581-585, or 612-619. In some embodiments, the sense strand consists of a sequence as set forth in any one of SEQ ID NOs: 1-192, 385-416, 467-517, 569-574, 581-585, or 612-619. In some embodiments, the sense strand comprises, or consists of, a sequence as set forth in any one of SEQ ID NOs: 385-416, 569-574, 581-585, 612-619.

One aspect of the present disclosure provides oligonucleotides for reducing expression of GYS2, in which the oligonucleotides comprise an antisense strand of 15 to 30 nucleotides in length. In some embodiments, the antisense strand has a region of complementarity to a target sequence of GYS2 as set forth in any one of SEQ ID NOs: 599-608. In some embodiments, the region of complementarity is at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, or at least 22 contiguous nucleotides in length. In some embodiments, the region of complementarity is fully complementary to the target sequence of GYS2. In some embodiments, the region of complementarity to GYS2 is at least 19 contiguous nucleotides in length.

In some embodiments, the antisense strand is 19 to 27 nucleotides in length. In some embodiments, the antisense strand is 21 to 27 nucleotides in length. In some embodiments, the oligonucleotide further comprises a sense strand of 15 to 40 nucleotides in length, in which the sense strand forms a duplex region with the antisense strand. In some embodiments, the sense strand is 19 to 40 nucleotides in length. In some embodiments, the antisense strand is 27 nucleotides in length and the sense strand is 25 nucleotides in length. In some embodiments, the duplex region is at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21 nucleotides in length. In some embodiments, the antisense strand and sense strand form a duplex region of 25 nucleotides in length.

In some embodiments, an oligonucleotide comprises an antisense strand and a sense strand that are each in a range of 21 to 23 nucleotides in length. In some embodiments, an oligonucleotide comprises a duplex structure in a range of 19 to 21 nucleotides in length. In some embodiments, an oligonucleotide comprises a 3′-overhang sequence of one or more nucleotides in length, in which the 3′-overhang sequence is present on the antisense strand, the sense strand, or the antisense strand and sense strand. In some embodiments, an oligonucleotide further comprises a 3′-overhang sequence on the antisense strand of two nucleotides in length. In some embodiments, an oligonucleotide comprises a 3′-overhang sequence of two nucleotides in length, in which the 3′-overhang sequence is present on the antisense strand, and in which the sense strand is 21 nucleotides in length and the antisense strand is 23 nucleotides in length, such that the sense strand and antisense strand form a duplex of 21 nucleotides in length.

In some embodiments, the sense strand comprises a sequence as set forth in any one of SEQ ID NOs: 1-192, 385-416, 467-517, 569-574, 581-585, or 612-619. In some embodiments, the sense strand consists of a sequence as set forth in any one of SEQ ID NOs: 1-192, 385-416, 467-517, 569-574, 581-585, or 612-619. In some embodiments, the antisense strand comprises a sequence as set forth in any one of SEQ ID NOs: 193-384, 417-466, 518-568, 575-580, 586-598, or 620-627. In some embodiments, the antisense strand consists of a sequence as set forth in any one of SEQ ID NOs: 193-384, 417-466, 518-568, 575-580, 586-598, or 620-627.

In some embodiments, the sense strand comprises at its 3′-end a stem-loop set forth as: S1-L-S2, in which S1 is complementary to S2, and in which L forms a loop between S1 and S2 of 3 to 5 nucleotides in length.

Another aspect of the present disclosure provides an oligonucleotide for reducing expression of GYS2, the oligonucleotide comprising an antisense strand and a sense strand, in which the antisense strand is 21 to 27 nucleotides in length and has a region of complementarity to GYS2, in which the sense strand comprises at its 3′-end a stem-loop set forth as: S1-L-S2, in which S1 is complementary to S2, and in which L forms a loop between S1 and S2 of 3 to 5 nucleotides in length, and in which the antisense strand and the sense strand form a duplex structure of at least 19 nucleotides in length but are not covalently linked (see, e.g., FIG. 3 ). In some embodiments, the region of complementarity is fully complementary to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21 contiguous nucleotides of GYS2 mRNA. In some embodiments, L is a tetraloop. In some embodiments, L is 4 nucleotides in length. In some embodiments, L comprises a sequence set forth as GAAA.

In some embodiments, an oligonucleotide comprises at least one modified nucleotide. In some embodiments, the modified nucleotide comprises a 2′-modification. In some embodiments, the 2′-modification is a modification selected from: 2′-aminoethyl, 2′-fluoro, 2′-O-methyl, 2′-O-methoxyethyl, and 2′-deoxy-2′-fluoro-β-d-arabinonucleic acid. In some embodiments, all of the nucleotides of an oligonucleotide are modified.

In some embodiments, an oligonucleotide comprises at least one modified internucleotide linkage. In some embodiments, the at least one modified internucleotide linkage is a phosphorothioate linkage. In some embodiments, the 4′-carbon of the sugar of the 5′-nucleotide of the antisense strand comprises a phosphate analog. In some embodiments, the phosphate analog is oxymethylphosphonate, vinylphosphonate, or malonylphosphonate.

In some embodiments, at least one nucleotide of an oligonucleotide is conjugated to one or more targeting ligands. In some embodiments, each targeting ligand comprises a carbohydrate, amino sugar, cholesterol, polypeptide, or lipid. In some embodiments, each targeting ligand comprises a N-acetylgalactosamine (GalNAc) moiety. In some embodiments, the GalNac moiety is a monovalent GalNAc moiety, a bivalent GalNAc moiety, a trivalent GalNAc moiety, or a tetravalent GalNAc moiety. In some embodiments, up to 4 nucleotides of L of the stem-loop are each conjugated to a monovalent GalNAc moiety. In some embodiments, the targeting ligand comprises an aptamer.

Another aspect of the present disclosure provides a composition comprising an oligonucleotide of the present disclosure and an excipient. Another aspect of the present disclosure provides a method comprising administering a composition of the present disclosure to a subject. In some embodiments, the method results in a decreased level or prevention of hepatomegaly, liver nodule formation, liver toxicity (e.g., levels of AST, ALT, and/or ALP), liver fibrosis, hepatocellular proliferation, fatty acid deposition in the liver, hepatic hyperplasia, hepatocellular adenoma, and/or hepatocellular carcinoma. Another aspect of the present disclosure provides a method for treating a glycogen storage disease or one or more symptoms of a glycogen storage disease. A non-limiting set of exemplary glycogen storage diseases may include: GSDI (e.g., GSDIa), GSDIII, GSDIV, GSDVI, and GSDIX.

Another aspect of the present disclosure provides an oligonucleotide for reducing expression of GYS2, the oligonucleotide comprising a sense strand of 15 to 40 nucleotides in length and an antisense strand of 15 to 30 nucleotides in length, in which the sense strand forms a duplex region with the antisense strand, in which the sense strand comprises a sequence as set forth in any one of SEQ ID NOs: 1-192, 385-416, 467-517, 569-574, 581-585, or 612-619, and results in a the antisense strand comprises a complementary sequence selected from SEQ ID NOs: 193-384, 417-466, 518-568, 575-580, 586-598, or 620-627.

In some embodiments, the oligonucleotide comprises a pair of sense and antisense strands selected from a row of the table set forth in Table 4.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate certain embodiments, and together with the written description, serve to provide non-limiting examples of certain aspects of the compositions and methods disclosed herein.

FIGS. 1A and 1B are graphs showing the percentage of GYS2 mRNA remaining after a screen of 264 GYS2 conjugates in HEK-293 cells. The nucleotide position in NM_021957.3 that corresponds to the 3′ end of the sense strand of each siRNA is indicated on the x axis.

FIGS. 2A and 2B is a set of graphs showing the percentage of mRNA remaining after GYS2 oligonucleotide screening of 71 GYS2 oligonucleotides at two or three different concentrations (0.1 nM and 1.0 nM or 0.03 nM, 0.1 nM, and 1.0 nM) in HEK-293 cells.

FIG. 3 is a schematic showing a non-limiting example of a double-stranded oligonucleotide with a nicked tetraloop structure that has been conjugated to four GalNAc moieties (diamond shapes).

FIG. 4 is a graph showing the results of screening in HEK-293 cells using GYS2 oligonucleotides of different base sequences in one or two different modification patterns. The X-axis lists the 3′ end of the sense strand targeted by the oligonucleotide evaluated. A negative control sequence (NCI), untransfected cells, and mock transfected cells are shown at left as controls.

FIG. 5 is a graph showing the results of screening in monkey hepatocyte cells using GYS2 oligonucleotides of different base sequences in the nicked tetraloop structure. The same modification pattern was used, and the oligonucleotides were tested at three different concentrations (0.1 μM, 0.3 μM, and 1.0 μM). Untransfected cells are shown as a control at left.

FIGS. 6A and 6B are a series of graphs showing the IC₅₀ results for GYS2 oligonucleotides selected from dose response curve screening in HEK-293 cells.

FIG. 7 is a graph showing an in vivo activity evaluation of GalNAc-conjugated GYS2 oligonucleotides in a nicked tetraloop structure. Eight different oligonucleotide sequences were tested. Oligonucleotides were subcutaneously administered to mice expressed human GYS2, at 0.5 mg/kg. The data show the amount of GYS2 mRNA remaining at day 4 following administration normalized to PBS control.

DETAILED DESCRIPTION OF THE INVENTION

According to some aspects, the disclosure provides oligonucleotides targeting GYS2 mRNA that are effective for reducing GYS2 expression in cells, particularly liver cells (e.g., hepatocytes) for the treatment of a glycogen storage disease (e.g., GSDIa, GSDIII, GSDIV, GSDVI, and GSDIX) or one or more symptoms of a glycogen storage disease. Accordingly, in related aspects, the disclosure provided methods of treating a glycogen storage disease (e.g., GSDIa, GSDIII, GSDIV, GSDVI, and GSDIX) or one or more symptoms of a glycogen storage disease that involve selectively reducing GYS2 gene expression in liver. In certain embodiments, GYS2 targeting oligonucleotides provided herein are designed for delivery to selected cells of target tissues (e.g., liver hepatocytes) to treat a glycogen storage disease (e.g., GSDIa, GSDIII, GSDIV, GSDVI, and GSDIX) or one or more symptoms of a glycogen storage disease in a subject.

Further aspects of the disclosure, including a description of defined terms, are provided below.

I. Definitions

Administering: As used herein, the terms “administering” or “administration” means to provide a substance (e.g., an oligonucleotide) to a subject in a manner that is pharmacologically useful (e.g., to treat a condition in the subject).

Approximately: As used herein, the term “approximately” or “about,” as applied to one or more values of interest, refers to a value that is similar to a stated reference value. In certain embodiments, the term “approximately” or “about” refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).

Asialoglycoprotein receptor (ASGPR): As used herein, the term “Asialoglycoprotein receptor” or “ASGPR” refers to a bipartite C-type lectin formed by a major 48 kDa (ASGPR-1) and minor 40 kDa subunit (ASGPR-2). ASGPR is primarily expressed on the sinusoidal surface of hepatocyte cells and has a major role in binding, internalization, and subsequent clearance of circulating glycoproteins that contain terminal galactose or N-acetylgalactosamine residues (asialoglycoproteins).

Complementary: As used herein, the term “complementary” refers to a structural relationship between nucleotides (e.g., two nucleotide on opposing nucleic acids or on opposing regions of a single nucleic acid strand) that permits the nucleotides to form base pairs with one another. For example, a purine nucleotide of one nucleic acid that is complementary to a pyrimidine nucleotide of an opposing nucleic acid may base pair together by forming hydrogen bonds with one another. In some embodiments, complementary nucleotides can base pair in the Watson-Crick manner or in any other manner that allows for the formation of stable duplexes. In some embodiments, two nucleic acids may have nucleotide sequences that are complementary to each other so as to form regions of complementarity, as described herein.

Deoxyribonucleotide: As used herein, the term “deoxyribonucleotide” refers to a nucleotide having a hydrogen at the 2′ position of its pentose sugar as compared with a ribonucleotide. A modified deoxyribonucleotide is a deoxyribonucleotide having one or more modifications or substitutions of atoms other than at the 2′ position, including modifications or substitutions in or of the sugar, phosphate group or base.

Double-stranded oligonucleotide: As used herein, the term “double-stranded oligonucleotide” refers to an oligonucleotide that is substantially in a duplex form. In some embodiments, complementary base-pairing of duplex region(s) of a double-stranded oligonucleotide is formed between antiparallel sequences of nucleotides of covalently separate nucleic acid strands. In some embodiments, complementary base-pairing of duplex region(s) of a double-stranded oligonucleotide is formed between antiparallel sequences of nucleotides of nucleic acid strands that are covalently linked. In some embodiments, complementary base-pairing of duplex region(s) of a double-stranded oligonucleotide is formed from a single nucleic acid strand that is folded (e.g., via a hairpin) to provide complementary antiparallel sequences of nucleotides that base pair together. In some embodiments, a double-stranded oligonucleotide comprises two covalently separate nucleic acid strands that are fully duplexed with one another. However, in some embodiments, a double-stranded oligonucleotide comprises two covalently separate nucleic acid strands that are partially duplexed, e.g., having overhangs at one or both ends. In some embodiments, a double-stranded oligonucleotide comprises antiparallel sequences of nucleotides that are partially complementary, and thus, may have one or more mismatches, which may include internal mismatches or end mismatches.

Duplex: As used herein, the term “duplex,” in reference to nucleic acids (e.g., oligonucleotides), refers to a structure formed through complementary base-pairing of two antiparallel sequences of nucleotides.

Excipient: As used herein, the term “excipient” refers to a non-therapeutic agent that may be included in a composition, for example, to provide or contribute to a desired consistency or stabilizing effect.

Glycogen Storage Disease: As used herein, the term “glycogen storage disease,” “GSD,” or “glycogen storage diseases” refers to metabolic disorders caused by enzyme deficiencies affecting glycogen synthesis, glycogen breakdown, and/or glucose breakdown (glycolysis). Various types of glycogen storage diseases have been characterized, including GSD 0, GSD I (also known as GSD 1 or von Gierke's disease; e.g., GSDIa), GSD II (also known as Pompe disease or acid maltase deficiency disease), GSD III (also known as GSD 3, Cori's disease, or Forbes' disease), GSD IV (GSD 4 or Andersen disease), GSD V (also known as McArdle disease), GSD VI (also known as GSD 6 or Hers' disease), GSD VII (also known as GSD 7 or Tarui's disease), GSD VIII, and GSD IX (also known as GSD 9). In some embodiments, individuals having a glycogen storage disease exhibit one or more of a number of symptoms including, but not limited to: hepatomegaly, increased liver toxicity (e.g., higher levels of AST, ALT, and/or ALP), liver fibrosis, fatty acid deposition in the liver, hepatic hyperplasia, hepatocellular adenoma, and/or hepatocellular carcinoma.

GYS2: as used herein, the term “GYS2” or “glycogen synthase 2” refers to the liver glycogen synthase gene. This gene encodes a protein, liver glycogen synthase, that catalyzes a rate-limiting stem in the synthesis of glycogen (i.e., the transfer of a glucose molecule from UDP-glucose to a terminal branch of the glycogen molecule). GYS2 is expressed in liver cells, e.g., hepatocytes. Homologs of GYS2 are conserved across a range of species, including human, mouse, rat, non-human primate species, and others (see, e.g., NCBI HomoloGene: 56580.) In humans, GYS2 encodes multiple transcripts, namely as set forth in GenBank accession numbers NM_021957.3 (SEQ ID NO: 609), XM_006719063.3, and XM_017019245.1, each encoding a different isoform, GenBank accession numbers NP_068776.2, XP_006719126.1 (isoform X1) and XP 016874734.1 (isoform X2), respectively. An example monkey (Rhesus macaque) transcript sequence is set forth in GenBank accession number XM_001098578.2 (SEQ ID NO: 610). An example mouse transcript is set forth in GenBank accession number NM_145572.2 (SEQ ID NO: 611).

Hepatocyte: As used herein, the term “hepatocyte” or “hepatocytes” refers to cells of the parenchymal tissues of the liver. These cells make up approximately 70-85% of the liver's mass and manufacture serum albumin, fibrinogen, and the prothrombin group of clotting factors (except for Factors 3 and 4). Markers for hepatocyte lineage cells may include, but are not limited to: transthyretin (Ttr), glutamine synthetase (Glul), hepatocyte nuclear factor 1a (Hnf1a), and hepatocyte nuclear factor 4a (Hnf4a). Markers for mature hepatocytes may include, but are not limited to: cytochrome P450 (Cyp3a11), fumarylacetoacetate hydrolase (Fah), glucose 6-phosphate (G6p), albumin (Alb), and OC2-2F8. See, e.g., Huch et al., (2013), Nature, 494(7436): 247-250, the contents of which relating to hepatocyte markers is incorporated herein by reference.

Loop: As used herein, the term “loop” refers to an unpaired region of a nucleic acid (e.g., oligonucleotide) that is flanked by two antiparallel regions of the nucleic acid that are sufficiently complementary to one another, such that under appropriate hybridization conditions (e.g., in a phosphate buffer, in a cells), the two antiparallel regions, which flank the unpaired region, hybridize to form a duplex (referred to as a “stem”).

Modified Internucleotide Linkage: As used herein, the term “modified internucleotide linkage” refers to an internucleotide linkage having one or more chemical modifications compared with a reference internucleotide linkage comprising a phosphodiester bond. In some embodiments, a modified nucleotide is a non-naturally occurring linkage. Typically, a modified internucleotide linkage confers one or more desirable properties to a nucleic acid in which the modified internucleotide linkage is present. For example, a modified nucleotide may improve thermal stability, resistance to degradation, nuclease resistance, solubility, bioavailability, bioactivity, reduced immunogenicity, etc.

Modified Nucleotide: As used herein, the term “modified nucleotide” refers to a nucleotide having one or more chemical modifications compared with a corresponding reference nucleotide selected from: adenine ribonucleotide, guanine ribonucleotide, cytosine ribonucleotide, uracil ribonucleotide, adenine deoxyribonucleotide, guanine deoxyribonucleotide, cytosine deoxyribonucleotide and thymidine deoxyribonucleotide. In some embodiments, a modified nucleotide is a non-naturally occurring nucleotide. In some embodiments, a modified nucleotide has one or more chemical modifications in its sugar, nucleobase and/or phosphate group. In some embodiments, a modified nucleotide has one or more chemical moieties conjugated to a corresponding reference nucleotide. Typically, a modified nucleotide confers one or more desirable properties to a nucleic acid in which the modified nucleotide is present. For example, a modified nucleotide may improve thermal stability, resistance to degradation, nuclease resistance, solubility, bioavailability, bioactivity, reduced immunogenicity, etc. In certain embodiments, a modified nucleotide comprises a 2′-O-methyl or a 2′-F substitution at the 2′ position of the ribose ring.

Nicked Tetraloop Structure: A “nicked tetraloop structure” is a structure of a RNAi oligonucleotide characterized by the presence of separate sense (passenger) and antisense (guide) strands, in which the sense strand has a region of complementarity to the antisense strand such that the two strands form a duplex, and in which at least one of the strands, generally the sense strand, extends from the duplex in which the extension contains a tetraloop and two self-complementary sequences forming a stem region adjacent to the tetraloop, in which the tetraloop is configured to stabilize the adjacent stem region formed by the self-complementary sequences of the at least one strand.

Oligonucleotide: As used herein, the term “oligonucleotide” refers to a short nucleic acid, e.g., of less than 100 nucleotides in length. An oligonucleotide can comprise ribonucleotides, deoxyribonucleotides, and/or modified nucleotides including, for example, modified ribonucleotides. An oligonucleotide may be single-stranded or double-stranded. An oligonucleotide may or may not have duplex regions. As a set of non-limiting examples, an oligonucleotide may be, but is not limited to, a small interfering RNA (siRNA), microRNA (miRNA), short hairpin RNA (shRNA), dicer substrate interfering RNA (dsiRNA), antisense oligonucleotide, short siRNA, or single-stranded siRNA. In some embodiments, a double-stranded oligonucleotide is an RNAi oligonucleotide.

Overhang: As used herein, the term “overhang” refers to terminal non-base-pairing nucleotide(s) resulting from one strand or region extending beyond the terminus of a complementary strand with which the one strand or region forms a duplex. In some embodiments, an overhang comprises one or more unpaired nucleotides extending from a duplex region at the 5′ terminus or 3′ terminus of a double-stranded oligonucleotide. In certain embodiments, the overhang is a 3′ or 5′ overhang on the antisense strand or sense strand of a double-stranded oligonucleotide.

Phosphate analog: As used herein, the term “phosphate analog” refers to a chemical moiety that mimics the electrostatic and/or steric properties of a phosphate group. In some embodiments, a phosphate analog is positioned at the 5′ terminal nucleotide of an oligonucleotide in place of a 5′-phosphate, which is often susceptible to enzymatic removal. In some embodiments, a 5′ phosphate analog contains a phosphatase-resistant linkage. Examples of phosphate analogs include 5′ phosphonates, such as 5′ methylenephosphonate (5′-MP) and 5′-(E)-vinylphosphonate (5′-VP). In some embodiments, an oligonucleotide has a phosphate analog at a 4′-carbon position of the sugar (referred to as a “4′-phosphate analog”) at a 5′-terminal nucleotide. An example of a 4′-phosphate analog is oxymethylphosphonate, in which the oxygen atom of the oxymethyl group is bound to the sugar moiety (e.g., at its 4′-carbon) or analog thereof. See, for example, International Patent Application PCT/US2017/049909, filed on Sep. 1, 2017, U.S. Provisional Application Nos. 62/383,207, filed on Sep. 2, 2016, and 62/393,401, filed on Sep. 12, 2016, the contents of each of which relating to phosphate analogs are incorporated herein by reference. Other modifications have been developed for the 5′ end of oligonucleotides (see, e.g., WO 2011/133871; U.S. Pat. No. 8,927,513; and Prakash et al. (2015), Nucleic Acids Res., 43(6):2993-3011, the contents of each of which relating to phosphate analogs are incorporated herein by reference).

Reduced expression: As used herein, the term “reduced expression” of a gene refers to a decrease in the amount of RNA transcript or protein encoded by the gene and/or a decrease in the amount of activity of the gene in a cell or subject, as compared to an appropriate reference cell or subject. For example, the act of treating a cell with a double-stranded oligonucleotide (e.g., one having an antisense strand that is complementary to GYS2 mRNA sequence) may result in a decrease in the amount of RNA transcript, protein and/or enzymatic activity (e.g., encoded by the GYS2 gene) compared to a cell that is not treated with the double-stranded oligonucleotide. Similarly, “reducing expression” as used herein refers to an act that results in reduced expression of a gene (e.g., GYS2).

Region of Complementarity: As used herein, the term “region of complementarity” refers to a sequence of nucleotides of a nucleic acid (e.g., a double-stranded oligonucleotide) that is sufficiently complementary to an antiparallel sequence of nucleotides (e.g., a target nucleotide sequence within an mRNA) to permit hybridization between the two sequences of nucleotides under appropriate hybridization conditions, e.g., in a phosphate buffer, in a cell, etc. A region of complementarity may be fully complementary to a nucleotide sequence (e.g., a target nucleotide sequence present within an mRNA or portion thereof). For example, a region of complementary that is fully complementary to a nucleotide sequence present in an mRNA has a contiguous sequence of nucleotides that is complementary, without any mismatches or gaps, to a corresponding sequence in the mRNA. Alternatively, a region of complementarity may be partially complementary to a nucleotide sequence (e.g., a nucleotide sequence present in an mRNA or portion thereof). For example, a region of complementary that is partially complementary to a nucleotide sequence present in an mRNA has a contiguous sequence of nucleotides that is complementary to a corresponding sequence in the mRNA but that contains one or more mismatches or gaps (e.g., 1, 2, 3, or more mismatches or gaps) compared with the corresponding sequence in the mRNA, provided that the region of complementarity remains capable of hybridizing with the mRNA under appropriate hybridization conditions.

Ribonucleotide: As used herein, the term “ribonucleotide” refers to a nucleotide having a ribose as its pentose sugar, which contains a hydroxyl group at its 2′ position. A modified ribonucleotide is a ribonucleotide having one or more modifications or substitutions of atoms other than at the 2′ position, including modifications or substitutions in or of the ribose, phosphate group or base.

RNAi Oligonucleotide: As used herein, the term “RNAi oligonucleotide” refers to either (a) a double stranded oligonucleotide having a sense strand (passenger) and antisense strand (guide), in which the antisense strand or part of the antisense strand is used by the Argonaute 2 (Ago2) endonuclease in the cleavage of a target mRNA or (b) a single stranded oligonucleotide having a single antisense strand, where that antisense strand (or part of that antisense strand) is used by the Ago2 endonuclease in the cleavage of a target mRNA.

Strand: As used herein, the term “strand” refers to a single contiguous sequence of nucleotides linked together through internucleotide linkages (e.g., phosphodiester linkages, phosphorothioate linkages). In some embodiments, a strand has two free ends, e.g., a 5′-end and a 3′-end.

Subject: As used herein, the term “subject” means any mammal, including mice, rabbits, and humans. In one embodiment, the subject is a human or non-human primate. The terms “individual” or “patient” may be used interchangeably with “subject.”

Synthetic: As used herein, the term “synthetic” refers to a nucleic acid or other molecule that is artificially synthesized (e.g., using a machine (e.g., a solid state nucleic acid synthesizer)) or that is otherwise not derived from a natural source (e.g., a cell or organism) that normally produces the molecule.

Targeting ligand: As used herein, the term “targeting ligand” refers to a molecule (e.g., a carbohydrate, amino sugar, cholesterol, polypeptide or lipid) that selectively binds to a cognate molecule (e.g., a receptor) of a tissue or cell of interest and that is conjugatable to another substance for purposes of targeting the other substance to the tissue or cell of interest. For example, in some embodiments, a targeting ligand may be conjugated to an oligonucleotide for purposes of targeting the oligonucleotide to a specific tissue or cell of interest. In some embodiments, a targeting ligand selectively binds to a cell surface receptor. Accordingly, in some embodiments, a targeting ligand when conjugated to an oligonucleotide facilitates delivery of the oligonucleotide into a particular cell through selective binding to a receptor expressed on the surface of the cell and endosomal internalization by the cell of the complex comprising the oligonucleotide, targeting ligand and receptor. In some embodiments, a targeting ligand is conjugated to an oligonucleotide via a linker that is cleaved following or during cellular internalization such that the oligonucleotide is released from the targeting ligand in the cell.

Tetraloop: As used herein, the term “tetraloop” refers to a loop that increases stability of an adjacent duplex formed by hybridization of flanking sequences of nucleotides. The increase in stability is detectable as an increase in melting temperature (T_(m)) of an adjacent stem duplex that is higher than the T_(m) of the adjacent stem duplex expected, on average, from a set of loops of comparable length consisting of randomly selected sequences of nucleotides. For example, a tetraloop can confer a melting temperature of at least 50° C., at least 55° C., at least 56° C., at least 58° C., at least 60° C., at least 65° C., or at least 75° C. in 10 mM NaHPO₄ to a hairpin comprising a duplex of at least 2 base pairs in length. In some embodiments, a tetraloop may stabilize a base pair in an adjacent stem duplex by stacking interactions. In addition, interactions among the nucleotides in a tetraloop include, but are not limited to: non-Watson-Crick base-pairing, stacking interactions, hydrogen bonding, and contact interactions (Cheong et al., Nature 1990 Aug. 16; 346(6285):680-2; Heus and Pardi, Science 1991 Jul. 12; 253(5016):191-4). In some embodiments, a tetraloop comprises or consists of 3 to 6 nucleotides, and is typically 4 to 5 nucleotides. In certain embodiments, a tetraloop comprises or consists of three, four, five, or six nucleotides, which may or may not be modified (e.g., which may or may not be conjugated to a targeting moiety). In one embodiment, a tetraloop consists of four nucleotides. Any nucleotide may be used in the tetraloop and standard IUPAC-IUB symbols for such nucleotides may be used as described in Cornish-Bowden (1985) Nucl. Acids Res. 13: 3021-3030. For example, the letter “N” may be used to mean that any base may be in that position, the letter “R” may be used to show that A (adenine) or G (guanine) may be in that position, and “B” may be used to show that C (cytosine), G (guanine), or T (thymine) may be in that position. Examples of tetraloops include the UNCG family of tetraloops (e.g., UUCG), the GNRA family of tetraloops (e.g., GAAA), and the CUUG tetraloop (Woese et al., Proc Natl Acad Sci USA. 1990 November; 87(21):8467-71; Antao et al., Nucleic Acids Res. 1991 Nov. 11; 19(21):5901-5). Examples of DNA tetraloops include the d(GNNA) family of tetraloops (e.g., d(GTTA)), the d(GNRA) family of tetraloops, the d(GNAB) family of tetraloops, the d(CNNG) family of tetraloops, and the d(TNCG) family of tetraloops (e.g., d(TTCG)). See, for example: Nakano et al. Biochemistry, 41 (48), 14281-14292, 2002. SHINJI et al. Nippon Kagakkai Koen Yokoshu VOL. 78th; NO. 2; PAGE. 731 (2000), which are incorporated by reference herein for their relevant disclosures. In some embodiments, the tetraloop is contained within a nicked tetraloop structure.

Treat: As used herein, the term “treat” refers to the act of providing care to a subject in need thereof, e.g., through the administration a therapeutic agent (e.g., an oligonucleotide) to the subject, for purposes of improving the health and/or well-being of the subject with respect to an existing condition (e.g., a disease, disorder) or to prevent or decrease the likelihood of the occurrence of a condition. In some embodiments, treatment involves reducing the frequency or severity of at least one sign, symptom or contributing factor of a condition (e.g., disease, disorder) experienced by a subject.

II. Oligonucleotide-Based Inhibitors

i. GYS2 Targeting Oligonucleotides

Potent oligonucleotides have been identified herein through examination of the GYS2 mRNA, including mRNAs of different species (human and Rhesus macaque, (see, e.g., Example 1)) and in vitro and in vivo testing. Such oligonucleotides can be used to achieve therapeutic benefit for subjects with a glycogen storage disease (e.g., GSDIa, GSDIII, GSDIV, GSDVI, and GSDIX) or one or more symptoms of a glycogen storage disease by reducing GYS2 activity, and consequently, by decreasing or preventing hepatomegaly, liver toxicity (demonstrated, e.g., levels of AST, ALT, and/or ALP), liver fibrosis, fatty acid deposition in the liver, hepatic hyperplasia, hepatocellular adenoma, and/or hepatocellular carcinoma. For example, potent RNAi oligonucleotides are provided herein that have a sense strand comprising, or consisting of, a sequence as set forth in any one of SEQ ID NOs: 1-192, 385-416, 467-517, 569-574, 581-585, or 612-619, and an antisense strand comprising, or consisting of, a complementary sequence selected from SEQ ID NOs: 193-384, 417-466, 518-568, 575-580, 586-598, or 620-627, as is also arranged the table provided in Table 4 (e.g., a sense strand comprising a sequence as set forth in SEQ ID NO: 1 and an antisense strand comprising a sequence as set forth in SEQ ID NO: 193).

The sequences can be put into multiple different oligonucleotide structures (or formats) as described herein.

In some embodiments, it has been discovered that certain regions of GYS2 mRNA are hotspots for targeting because they are more amenable than other regions to oligonucleotide-based inhibition. In some embodiments, a hotspot region of GYS2 consists of a sequence as forth in any one of SEQ ID NOs: 599-608. These regions of GYS2 mRNA may be targeted using oligonucleotides as discussed herein for purposes of inhibiting GYS2 mRNA expression.

Accordingly, in some embodiments, oligonucleotides provided herein are designed so as to have regions of complementarity to GYS2 mRNA (e.g., within a hotspot of GYS2 mRNA) for purposes of targeting the mRNA in cells and inhibiting its expression. The region of complementarity is generally of a suitable length and base content to enable annealing of the oligonucleotide (or a strand thereof) to GYS2 mRNA for purposes of inhibiting its expression.

In some embodiments, an oligonucleotide disclosed herein comprises a region of complementarity (e.g., on an antisense strand of a double-stranded oligonucleotide) that is at least partially complementary to a sequence as set forth in SEQ ID NOs: 1-192, 385-416, 467-517, 569-574, 581-585, or 612-619, which include sequences mapping to within hotspot regions of GYS2 mRNA. In some embodiments, an oligonucleotide disclosed herein comprises a region of complementarity (e.g., on an antisense strand of a double-stranded oligonucleotide) that is fully complementary to a sequence as set forth in SEQ ID NOs: 1-192, 385-416, 467-517, 569-574, 581-585, or 612-619. In some embodiments, a region of complementarity of an oligonucleotide that is complementary to contiguous nucleotides of a sequence as set forth in SEQ ID NOs: 1-192, 385-416, 467-517, 569-574, 581-585, or 612-619 spans the entire length of an antisense strand. In some embodiments, a region of complementarity of an oligonucleotide that is complementary to contiguous nucleotides of a sequence as set forth in any one of SEQ ID NOs: 1-192, 385-416, 467-517, 569-574, 581-585, or 612-619 spans a portion of the entire length of an antisense strand (e.g., all but two nucleotides at the 3′ end of the antisense strand). In some embodiments, an oligonucleotide disclosed herein comprises a region of complementarity (e.g., on an antisense strand of a double-stranded oligonucleotide) that is at least partially (e.g., fully) complementary to a contiguous stretch of nucleotides spanning nucleotides 1-19 of a sequence as set forth in SEQ ID NOs: 1-192, 385-416, 467-517, 569-574, 581-585, or 612-619.

In some embodiments, the region of complementarity is at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 nucleotides in length. In some embodiments, an oligonucleotide provided herein has a region of complementarity to GYS2 mRNA that is in the range of 12 to 30 (e.g., 12 to 30, 12 to 22, 15 to 25, 17 to 21, 18 to 27, 19 to 27, or 15 to 30) nucleotides in length. In some embodiments, an oligonucleotide provided herein has a region of complementarity to GYS2 mRNA that is 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.

In some embodiments, a region of complementarity to GYS2 mRNA may have one or more mismatches compared with a corresponding sequence of GYS2 mRNA. A region of complementarity on an oligonucleotide may have up to 1, up to 2, up to 3, up to 4 etc. mismatches provided that it maintains the ability to form complementary base pairs with GYS2 mRNA under appropriate hybridization conditions. Alternatively, a region of complementarity on an oligonucleotide may have no more than 1, no more than 2, no more than 3, or no more than 4 mismatches provided that it maintains the ability to form complementary base pairs with GYS2 mRNA under appropriate hybridization conditions. In some embodiments, if there are more than one mismatches in a region of complementarity, they may be positioned consecutively (e.g., 2, 3, 4, or more in a row), or interspersed throughout the region of complementarity provided that the oligonucleotide maintains the ability to form complementary base pairs with GYS2 mRNA under appropriate hybridization conditions.

Still, in some embodiments, double-stranded oligonucleotides provided herein comprise, or consist of, a sense strand having a sequence as set forth in any one of SEQ ID NOs: 1-192, 385-416, 467-517, 569-574, 581-585, or 612-619 and an antisense strand having a complementary sequence selected from SEQ ID NOs: 193-384, 417-466, 518-568, 575-580, 586-598, or 620-627, as is arranged in the table provided in Table 4 (e.g., a sense strand comprising a sequence as set forth in SEQ ID NO: 1 and an antisense strand comprising a sequence as set forth in SEQ ID NO: 193).

ii. Oligonucleotide Structures

There are a variety of structures of oligonucleotides that are useful for targeting GYS2 mRNA in the methods of the present disclosure, including RNAi, miRNA, etc. Any of the structures described herein or elsewhere may be used as a framework to incorporate or target a sequence described herein (e.g., a hotpot sequence of GYS2 such as those illustrated in SEQ ID NOs: 599-608, or a sense or antisense strand that comprises or consists of a sequence as set forth SEQ ID NOs: 1-192, 385-416, 467-517, 569-574, 581-585, or 612-619 or as set forth SEQ ID NOs: 193-384, 417-466, 518-568, 575-580, 586-598, or 620-627, respectively). Double-stranded oligonucleotides for targeting GYS2 expression (e.g., via the RNAi pathway) generally have a sense strand and an antisense strand that form a duplex with one another. In some embodiments, the sense and antisense strands are not covalently linked. However, in some embodiments, the sense and antisense strands are covalently linked.

In some embodiments, sequences described herein can be incorporated into, or targeted using, oligonucleotides that comprise sense and antisense strands that are both in the range of 17 to 40 nucleotides in length. In some embodiments, oligonucleotides incorporating such sequences are provided that have a tetraloop structure within a 3′ extension of their sense strand, and two terminal overhang nucleotides at the 3′ end of its antisense strand. In some embodiments, the two terminal overhang nucleotides are GG. Typically, one or both of the two terminal GG nucleotides of the antisense strand is or are not complementary to the target.

In some embodiments, oligonucleotides incorporating such sequences are provided that have sense and antisense strands that are both in the range of 21 to 23 nucleotides in length. In some embodiments, a 3′ overhang is provided on the sense, antisense, or both sense and antisense strands that is 1 or 2 nucleotides in length. In some embodiments, an oligonucleotide has a guide strand of 23 nucleotides and a passenger strand of 21 nucleotides, in which the 3′-end of passenger strand and 5′-end of guide strand form a blunt end and where the guide strand has a two nucleotide 3′ overhang.

In some embodiments, double-stranded oligonucleotides for reducing GYS2 expression engage RNA interference (RNAi). For example, RNAi oligonucleotides have been developed with each strand having sizes of 19-25 nucleotides with at least one 3′ overhang of 1 to 5 nucleotides (see, e.g., U.S. Pat. No. 8,372,968). Longer oligonucleotides have also been developed that are processed by Dicer to generate active RNAi products (see, e.g., U.S. Pat. No. 8,883,996). Further work produced extended double-stranded oligonucleotides where at least one end of at least one strand is extended beyond a duplex targeting region, including structures where one of the strands includes a thermodynamically-stabilizing tetraloop structure (see, e.g., U.S. Pat. Nos. 8,513,207 and 8,927,705, as well as WO2010033225, which are incorporated by reference herein for their disclosure of these oligonucleotides). Such structures may include single-stranded extensions (on one or both sides of the molecule) as well as double-stranded extensions.

In some embodiments, oligonucleotides may be in the range of 21 to 23 nucleotides in length. In some embodiments, oligonucleotides may have an overhang (e.g., of 1, 2, or 3 nucleotides in length) in the 3′ end of the sense and/or antisense strands. In some embodiments, oligonucleotides (e.g., siRNAs) may comprise a 21 nucleotide guide strand that is antisense to a target RNA and a complementary passenger strand, in which both strands anneal to form a 19-bp duplex and 2 nucleotide overhangs at either or both 3′ ends. See, for example, U.S. Pat. Nos. 9,012,138, 9,012,621, and 9,193,753, the contents of each of which are incorporated herein for their relevant disclosures.

In some embodiments, an oligonucleotide of the invention has a 36 nucleotide sense strand that comprises an region extending beyond the antisense-sense duplex, where the extension region has a stem-tetraloop structure where the stem is a six base pair duplex and where the tetraloop has four nucleotides. In some embodiments, the stem-tetraloop is set forth as: S₁-L-S₂, in which S₁ is complementary to S₂ so as to form a duplex, and in which L forms a tetraloop between S₁ and S₂.

In certain of those embodiments, three or four of the tetraloop nucleotides are each conjugated to a monovalent GalNac ligand.

In some embodiments, an oligonucleotide of the invention comprises a 25 nucleotide sense strand and a 27 nucleotide antisense strand that when acted upon by a dicer enzyme results in an antisense strand that is incorporated into the mature RISC.

Other oligonucleotides designs for use with the compositions and methods disclosed herein include: 16-mer siRNAs (see, e.g., Nucleic Acids in Chemistry and Biology. Blackburn (ed.), Royal Society of Chemistry, 2006), shRNAs (e.g., having 19 bp or shorter stems; see, e.g., Moore et al. Methods Mol. Biol. 2010; 629:141-158), blunt siRNAs (e.g., of 19 bps in length; see: e.g., Kraynack and Baker, RNA Vol. 12, p 163-176 (2006)), asymmetrical siRNAs (aiRNA; see, e.g., Sun et al., Nat. Biotechnol. 26, 1379-1382 (2008)), asymmetric shorter-duplex siRNA (see, e.g., Chang et al., Mol Ther. 2009 April; 17(4): 725-32), fork siRNAs (see, e.g., Hohjoh, FEBS Letters, Vol 557, issues 1-3; January 2004, p 193-198), single-stranded siRNAs (Elsner; Nature Biotechnology 30, 1063 (2012)), dumbbell-shaped circular siRNAs (see, e.g., Abe et al. J Am Chem Soc 129: 15108-15109 (2007)), and small internally segmented interfering RNA (sisiRNA; see, e.g., Bramsen et al., Nucleic Acids Res. 2007 September; 35(17): 5886-5897). Each of the foregoing references is incorporated by reference in its entirety for the related disclosures therein. Further non-limiting examples of an oligonucleotide structures that may be used in some embodiments to reduce or inhibit the expression of GYS2 are microRNA (miRNA), short hairpin RNA (shRNA), and short siRNA (see, e.g., Hamilton et al., Embo J., 2002, 21(17): 4671-4679; see also U.S. Application No. 20090099115).

a. Antisense Strands

In some embodiments, an oligonucleotide disclosed herein for targeting GYS2 comprises an antisense strand comprising or consisting of a sequence as set forth in any one of SEQ ID NOs: 193-384, 417-466, 518-568, 575-580, 586-598, or 620-627. In some embodiments, an oligonucleotide comprises an antisense strand comprising or consisting of at least 12 (e.g., at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, or at least 23) contiguous nucleotides of a sequence as set forth in any one of SEQ ID NOs: 193-384, 417-466, 518-568, 575-580, 586-598, or 620-627.

In some embodiments, a double-stranded oligonucleotide may have an antisense strand of up to 40 nucleotides in length (e.g., up to 40, up to 35, up to 30, up to 27, up to 25, up to 21, up to 19, up to 17, or up to 12 nucleotides in length). In some embodiments, an oligonucleotide may have an antisense strand of at least 12 nucleotides in length (e.g., at least 12, at least 15, at least 19, at least 21, at least 22, at least 25, at least 27, at least 30, at least 35, or at least 38 nucleotides in length). In some embodiments, an oligonucleotide may have an antisense strand in a range of 12 to 40 (e.g., 12 to 40, 12 to 36, 12 to 32, 12 to 28, 15 to 40, 15 to 36, 15 to 32, 15 to 28, 17 to 22, 17 to 25, 19 to 27, 19 to 30, 20 to 40, 22 to 40, 25 to 40, or 32 to 40) nucleotides in length. In some embodiments, an oligonucleotide may have an antisense strand of 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides in length.

In some embodiments, an antisense strand of an oligonucleotide may be referred to as a “guide strand.” For example, if an antisense strand can engage with RNA-induced silencing complex (RISC) and bind to an Argonaut protein, or engage with or bind to one or more similar factors, and direct silencing of a target gene, it may be referred to as a guide strand. In some embodiments, a sense strand complementary to a guide strand may be referred to as a “passenger strand.”

b. Sense Strands

In some embodiments, an oligonucleotide disclosed herein for targeting GYS2 comprises or consists of a sense strand sequence as set forth in in any one of SEQ ID NOs: 1-192, 385-416, 467-517, 569-574, 581-585, or 612-619. In some embodiments, an oligonucleotide has a sense strand that comprises or consists of at least 12 (e.g., at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, or at least 23) contiguous nucleotides of a sequence as set forth in in any one of SEQ ID NOs: 1-192, 385-416, 467-517, 569-574, 581-585, or 612-619.

In some embodiments, an oligonucleotide may have a sense strand (or passenger strand) of up to 40 nucleotides in length (e.g., up to 40, up to 36, up to 30, up to 27, up to 25, up to 21, up to 19, up to 17, or up to 12 nucleotides in length). In some embodiments, an oligonucleotide may have a sense strand of at least 12 nucleotides in length (e.g., at least 12, at least 15, at least 19, at least 21, at least 25, at least 27, at least 30, at least 36, or at least 38 nucleotides in length). In some embodiments, an oligonucleotide may have a sense strand in a range of 12 to 40 (e.g., 12 to 40, 12 to 36, 12 to 32, 12 to 28, 15 to 40, 15 to 36, 15 to 32, 15 to 28, 17 to 21, 17 to 25, 19 to 27, 19 to 30, 20 to 40, 22 to 40, 25 to 40, or 32 to 40) nucleotides in length. In some embodiments, an oligonucleotide may have a sense strand of 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides in length.

In some embodiments, a sense strand comprises a stem-loop structure at its 3′-end. In some embodiments, a sense strand comprises a stem-loop structure at its 5′-end. In some embodiments, a stem is a duplex of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 base pairs in length. In some embodiments, a stem-loop provides the molecule better protection against degradation (e.g., enzymatic degradation) and facilitates targeting characteristics for delivery to a target cell. For example, in some embodiments, a loop provides added nucleotides on which modification can be made without substantially affecting the gene expression inhibition activity of an oligonucleotide. In certain embodiments, an oligonucleotide is provided herein in which the sense strand comprises (e.g., at its 3′-end) a stem-loop set forth as: S₁-L-S₂, in which S₁ is complementary to S₂, and in which L forms a loop between S₁ and S₂ of up to 10 nucleotides in length (e.g., 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length).

In some embodiments, a loop (L) of a stem-loop is a tetraloop (e.g., within a nicked tetraloop structure). A tetraloop may contain ribonucleotides, deoxyribonucleotides, modified nucleotides, and combinations thereof. Typically, a tetraloop has 4 to 5 nucleotides.

c. Duplex Length

In some embodiments, a duplex formed between a sense and antisense strand is at least 12 (e.g., at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21) nucleotides in length. In some embodiments, a duplex formed between a sense and antisense strand is in the range of 12-30 nucleotides in length (e.g., 12 to 30, 12 to 27, 12 to 22, 15 to 25, 18 to 30, 18 to 22, 18 to 25, 18 to 27, 18 to 30, 19 to 30, or 21 to 30 nucleotides in length). In some embodiments, a duplex formed between a sense and antisense strand is 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. In some embodiments a duplex formed between a sense and antisense strand does not span the entire length of the sense strand and/or antisense strand. In some embodiments, a duplex between a sense and antisense strand spans the entire length of either the sense or antisense strands. In certain embodiments, a duplex between a sense and antisense strand spans the entire length of both the sense strand and the antisense strand.

d. Oligonucleotide Ends

In some embodiments, an oligonucleotide provided herein comprises sense and antisense strands, such that there is a 3′-overhang on either the sense strand or the antisense strand, or both the sense and antisense strand. In some embodiments, oligonucleotides provided herein have one 5′end that is thermodynamically less stable compared to the other 5′ end. In some embodiments, an asymmetric oligonucleotide is provided that includes a blunt end at the 3′ end of a sense strand and an overhang at the 3′ end of an antisense strand. In some embodiments, a 3′ overhang on an antisense strand is 1-8 nucleotides in length (e.g., 1, 2, 3, 4, 5, 6, 7, or 8 nucleotides in length).

Typically, an oligonucleotide for RNAi has a two nucleotide overhang on the 3′ end of the antisense (guide) strand. However, other overhangs are possible. In some embodiments, an overhang is a 3′ overhang comprising a length of between one and six nucleotides, optionally one to five, one to four, one to three, one to two, two to six, two to five, two to four, two to three, three to six, three to five, three to four, four to six, four to five, five to six nucleotides, or one, two, three, four, five or six nucleotides. However, in some embodiments, the overhang is a 5′ overhang comprising a length of between one and six nucleotides, optionally one to five, one to four, one to three, one to two, two to six, two to five, two to four, two to three, three to six, three to five, three to four, four to six, four to five, five to six nucleotides, or one, two, three, four, five or six nucleotides.

In some embodiments, one or more (e.g., 2, 3, 4) terminal nucleotides of the 3′ end or 5′ end of a sense and/or antisense strand are modified. For example, in some embodiments, one or two terminal nucleotides of the 3′ end of an antisense strand are modified. In some embodiments, the last nucleotide at the 3′ end of an antisense strand is modified, e.g., comprises 2′-modification, e.g., a 2′-O-methoxyethyl. In some embodiments, the last one or two terminal nucleotides at the 3′ end of an antisense strand are complementary to the target. In some embodiments, the last one or two nucleotides at the 3′ end of the antisense strand are not complementary to the target. In some embodiments, the 5′ end and/or the 3′ end of a sense or antisense strand has an inverted cap nucleotide.

e. Mismatches

In some embodiments, there is one or more (e.g., 1, 2, 3, or 4) mismatches between a sense and antisense strand. If there is more than one mismatch between a sense and antisense strand, they may be positioned consecutively (e.g., 2, 3 or more in a row), or interspersed throughout the region of complementarity. In some embodiments, the 3′-terminus of the sense strand contains one or more mismatches. In one embodiment, two mismatches are incorporated at the 3′ terminus of the sense strand. In some embodiments, base mismatches or destabilization of segments at the 3′-end of the sense strand of the oligonucleotide improved the potency of synthetic duplexes in RNAi, possibly through facilitating processing by Dicer.

iii. Single-Stranded Oligonucleotides

In some embodiments, an oligonucleotide for reducing GYS2 expression as described herein is single-stranded. Such structures may include, but are not limited to single-stranded RNAi oligonucleotides. Recent efforts have demonstrated the activity of single-stranded RNAi oligonucleotides (see, e.g., Matsui et al. (May 2016), Molecular Therapy, Vol. 24(5), 946-955). However, in some embodiments, oligonucleotides provided herein are antisense oligonucleotides (ASOs). An antisense oligonucleotide is a single-stranded oligonucleotide that has a nucleobase sequence which, when written in the 5′ to 3′ direction, comprises the reverse complement of a targeted segment of a particular nucleic acid and is suitably modified (e.g., as a gapmer) so as to induce RNaseH mediated cleavage of its target RNA in cells or (e.g., as a mixmer) so as to inhibit translation of the target mRNA in cells. Antisense oligonucleotides for use in the instant disclosure may be modified in any suitable manner known in the art including, for example, as shown in U.S. Pat. No. 9,567,587, which is incorporated by reference herein for its disclosure regarding modification of antisense oligonucleotides (including, e.g., length, sugar moieties of the nucleobase (pyrimidine, purine), and alterations of the heterocyclic portion of the nucleobase). Further, antisense molecules have been used for decades to reduce expression of specific target genes (see, e.g., Bennett et al.; Pharmacology of Antisense Drugs, Annual Review of Pharmacology and Toxicology, Vol. 57: 81-105).

iv. Oligonucleotide Modifications

Oligonucleotides may be modified in various ways to improve or control specificity, stability, delivery, bioavailability, resistance from nuclease degradation, immunogenicity, base-paring properties, RNA distribution and cellular uptake and other features relevant to therapeutic or research use. See, e.g., Bramsen et al., Nucleic Acids Res., 2009, 37, 2867-2881; Bramsen and Kjems (Frontiers in Genetics, 3 (2012): 1-22). Accordingly, in some embodiments, oligonucleotides of the present disclosure may include one or more suitable modifications. In some embodiments, a modified nucleotide has a modification in its base (or nucleobase), the sugar (e.g., ribose, deoxyribose), or the phosphate group.

The number of modifications on an oligonucleotide and the positions of those nucleotide modifications may influence the properties of an oligonucleotide. For example, oligonucleotides may be delivered in vivo by conjugating them to or encompassing them in a lipid nanoparticle (LNP) or similar carrier. However, when an oligonucleotide is not protected by an LNP or similar carrier (e.g., “naked delivery”), it may be advantageous for at least some of the its nucleotides to be modified. Accordingly, in certain embodiments of any of the oligonucleotides provided herein, all or substantially all of the nucleotides of an oligonucleotide are modified. In certain embodiments, more than half of the nucleotides are modified. In certain embodiments, less than half of the nucleotides are modified. Typically, with naked delivery, every nucleotide is modified at the 2′-position of the sugar group of that nucleotide. These modifications may be reversible or irreversible. Typically, the 2′-position modification is 2′-fluoro, 2′-O-methyl, etc. In some embodiments, an oligonucleotide as disclosed herein has a number and type of modified nucleotides sufficient to cause the desired characteristic (e.g., protection from enzymatic degradation, capacity to target a desired cell after in vivo administration, and/or thermodynamic stability).

a. Sugar Modifications

In some embodiments, a modified sugar (also referred to herein as a sugar analog) includes a modified deoxyribose or ribose moiety, e.g., in which one or more modifications occur at the 2′, 3′, 4′, and/or 5′ carbon position of the sugar. In some embodiments, a modified sugar may also include non-natural alternative carbon structures such as those present in locked nucleic acids (“LNA”) (see, e.g., Koshkin et al. (1998), Tetrahedron 54, 3607-3630), unlocked nucleic acids (“UNA”) (see, e.g., Snead et al. (2013), Molecular Therapy—Nucleic Acids, 2, e103), and bridged nucleic acids (“BNA”) (see, e.g., Imanishi and Obika (2002), The Royal Society of Chemistry, Chem. Commun., 1653-1659). Koshkin et al., Snead et al., and Imanishi and Obika are incorporated by reference herein for their disclosures relating to sugar modifications.

In some embodiments, a nucleotide modification in a sugar comprises a 2′-modification. In certain embodiments, the 2′-modification may be 2′-aminoethyl, 2′-fluoro, 2′-O-methyl, 2′-O-methoxyethyl, or 2′-deoxy-2′-fluoro-β-d-arabinonucleic acid. Typically, the modification is 2′-fluoro, 2′-O-methyl, or 2′-O-methoxyethyl. However, a large variety of 2′ position modifications that have been developed for use in oligonucleotides can be employed in oligonucleotides disclosed herein. See, e.g., Bramsen et al., Nucleic Acids Res., 2009, 37, 2867-2881. In some embodiments, a modification in a sugar comprises a modification of the sugar ring, which may comprise modification of one or more carbons of the sugar ring. For example, a modification of a sugar of a nucleotide may comprise a linkage between the 2′-carbon and a 1′-carbon or 4′-carbon of the sugar. For example, the linkage may comprise an ethylene or methylene bridge. In some embodiments, a modified nucleotide has an acyclic sugar that lacks a 2′-carbon to 3′-carbon bond. In some embodiments, a modified nucleotide has a thiol group, e.g., in the 4′ position of the sugar.

In some embodiments, the terminal 3′-end group (e.g., a 3′-hydroxyl) is a phosphate group or other group, which can be used, for example, to attach linkers, adapters or labels or for the direct ligation of an oligonucleotide to another nucleic acid.

b. 5′ Terminal Phosphates

5′-terminal phosphate groups of oligonucleotides may or in some circumstances enhance the interaction with Argonaut 2. However, oligonucleotides comprising a 5′-phosphate group may be susceptible to degradation via phosphatases or other enzymes, which can limit their bioavailability in vivo. In some embodiments, oligonucleotides include analogs of 5′ phosphates that are resistant to such degradation. In some embodiments, a phosphate analog may be oxymethylphosphonate, vinylphosphonate, or malonylphosphonate. In certain embodiments, the 5′ end of an oligonucleotide strand is attached to a chemical moiety that mimics the electrostatic and steric properties of a natural 5′-phosphate group (“phosphate mimic”) (see, e.g., Prakash et al. (2015), Nucleic Acids Res., Nucleic Acids Res. 2015 Mar. 31; 43(6): 2993-3011, the contents of which relating to phosphate analogs are incorporated herein by reference). Many phosphate mimics have been developed that can be attached to the 5′ end (see, e.g., U.S. Pat. No. 8,927,513, the contents of which relating to phosphate analogs are incorporated herein by reference). Other modifications have been developed for the 5′ end of oligonucleotides (see, e.g., WO 2011/133871, the contents of which relating to phosphate analogs are incorporated herein by reference). In certain embodiments, a hydroxyl group is attached to the 5′ end of the oligonucleotide.

In some embodiments, an oligonucleotide has a phosphate analog at a 4′-carbon position of the sugar (referred to as a “4′-phosphate analog”). See, for example, International Patent Application PCT/US2017/049909, filed on Sep. 1, 2017, U.S. Provisional Application Nos. 62/383,207, entitled 4′-Phosphate Analogs and Oligonucleotides Comprising the Same, filed on Sep. 2, 2016, and 62/393,401, filed on Sep. 12, 2016, entitled 4′-Phosphate Analogs and Oligonucleotides Comprising the Same, the contents of each of which relating to phosphate analogs are incorporated herein by reference. In some embodiments, an oligonucleotide provided herein comprises a 4′-phosphate analog at a 5′-terminal nucleotide. In some embodiments, a phosphate analog is an oxymethylphosphonate, in which the oxygen atom of the oxymethyl group is bound to the sugar moiety (e.g., at its 4′-carbon) or analog thereof. In other embodiments, a 4′-phosphate analog is a thiomethylphosphonate or an aminomethylphosphonate, in which the sulfur atom of the thiomethyl group or the nitrogen atom of the aminomethyl group is bound to the 4′-carbon of the sugar moiety or analog thereof. In certain embodiments, a 4′-phosphate analog is an oxymethylphosphonate. In some embodiments, an oxymethylphosphonate is represented by the formula —O—CH₂—PO(OH)₂ or —O—CH₂—PO(OR)₂, in which R is independently selected from H, CH₃, an alkyl group, CH₂CH₂CN, CH₂OCOC(CH₃)₃, CH₂OCH₂CH₂Si(CH₃)₃, or a protecting group. In certain embodiments, the alkyl group is CH₂CH₃. More typically, R is independently selected from H, CH₃, or CH₂CH₃.

c. Modified Internucleoside Linkages

In some embodiments, the oligonucleotide may comprise a modified internucleoside linkage. In some embodiments, phosphate modifications or substitutions may result in an oligonucleotide that comprises at least one (e.g., at least 1, at least 2, at least 3, at least 4, or at least 5) modified internucleotide linkage. In some embodiments, any one of the oligonucleotides disclosed herein comprises 1 to 10 (e.g., 1 to 10, 2 to 8, 4 to 6, 3 to 10, 5 to 10, 1 to 5, 1 to 3 or 1 to 2) modified internucleotide linkages. In some embodiments, any one of the oligonucleotides disclosed herein comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 modified internucleotide linkages.

A modified internucleotide linkage may be a phosphorodithioate linkage, a phosphorothioate linkage, a phosphotriester linkage, a thionoalkylphosphonate linkage, a thionoalkylphosphotriester linkage, a phosphoramidite linkage, a phosphonate linkage or a boranophosphate linkage. In some embodiments, at least one modified internucleotide linkage of any one of the oligonucleotides as disclosed herein is a phosphorothioate linkage.

d. Base modifications

In some embodiments, oligonucleotides provided herein have one or more modified nucleobases. In some embodiments, modified nucleobases (also referred to herein as base analogs) are linked at the 1′ position of a nucleotide sugar moiety. In certain embodiments, a modified nucleobase is a nitrogenous base. In certain embodiments, a modified nucleobase does not contain a nitrogen atom. See e.g., U.S. Published Patent Application No. 20080274462. In some embodiments, a modified nucleotide comprises a universal base. However, in certain embodiments, a modified nucleotide does not contain a nucleobase (abasic).

In some embodiments, a universal base is a heterocyclic moiety located at the 1′ position of a nucleotide sugar moiety in a modified nucleotide, or the equivalent position in a nucleotide sugar moiety substitution that, when present in a duplex, can be positioned opposite more than one type of base without substantially altering the structure of the duplex. In some embodiments, compared to a reference single-stranded nucleic acid (e.g., oligonucleotide) that is fully complementary to a target nucleic acid, a single-stranded nucleic acid containing a universal base forms a duplex with the target nucleic acid that has a lower T_(m) than a duplex formed with the complementary nucleic acid. However, in some embodiments, compared to a reference single-stranded nucleic acid in which the universal base has been replaced with a base to generate a single mismatch, the single-stranded nucleic acid containing the universal base forms a duplex with the target nucleic acid that has a higher T_(m) than a duplex formed with the nucleic acid comprising the mismatched base.

Non-limiting examples of universal-binding nucleotides include inosine, 1-β-D-ribofuranosyl-5-nitroindole, and/or 1-β-D-ribofuranosyl-3-nitropyrrole (US Pat. Appl. Publ. No. 20070254362 to Quay et al.; Van Aerschot et al., An acyclic 5-nitroindazole nucleoside analogue as ambiguous nucleoside. Nucleic Acids Res. 1995 Nov. 11; 23(21):4363-70; Loakes et al., 3-Nitropyrrole and 5-nitroindole as universal bases in primers for DNA sequencing and PCR. Nucleic Acids Res. 1995 Jul. 11; 23(13):2361-6; Loakes and Brown, 5-Nitroindole as an universal base analogue. Nucleic Acids Res. 1994 Oct. 11; 22(20):4039-43. Each of the foregoing is incorporated by reference herein for their disclosures relating to base modifications).

e. Reversible Modifications

While certain modifications to protect an oligonucleotide from the in vivo environment before reaching target cells can be made, they can reduce the potency or activity of the oligonucleotide once it reaches the cytosol of the target cell. Reversible modifications can be made such that the molecule retains desirable properties outside of the cell, which are then removed upon entering the cytosolic environment of the cell. Reversible modification can be removed, for example, by the action of an intracellular enzyme or by the chemical conditions inside of a cell (e.g., through reduction by intracellular glutathione).

In some embodiments, a reversibly modified nucleotide comprises a glutathione-sensitive moiety. Typically, nucleic acid molecules have been chemically modified with cyclic disulfide moieties to mask the negative charge created by the internucleotide diphosphate linkages and improve cellular uptake and nuclease resistance. See U.S. Published Application No. 2011/0294869 originally assigned to Traversa Therapeutics, Inc. (“Traversa”), PCT Publication No. WO 2015/188197 to Solstice Biologics, Ltd. (“Solstice”), Meade et al., Nature Biotechnology, 2014, 32:1256-1263 (“Meade”), PCT Publication No. WO 2014/088920 to Merck Sharp & Dohme Corp, each of which are incorporated by reference for their disclosures of such modifications. This reversible modification of the internucleotide diphosphate linkages is designed to be cleaved intracellularly by the reducing environment of the cytosol (e.g. glutathione). Earlier examples include neutralizing phosphotriester modifications that were reported to be cleavable inside cells (Dellinger et al. J. Am. Chem. Soc. 2003, 125:940-950).

In some embodiments, such a reversible modification allows protection during in vivo administration (e.g., transit through the blood and/or lysosomal/endosomal compartments of a cell) where the oligonucleotide will be exposed to nucleases and other harsh environmental conditions (e.g., pH). When released into the cytosol of a cell where the levels of glutathione are higher compared to extracellular space, the modification is reversed and the result is a cleaved oligonucleotide. Using reversible, glutathione sensitive moieties, it is possible to introduce sterically larger chemical groups into the oligonucleotide of interest as compared to the options available using irreversible chemical modifications. This is because these larger chemical groups will be removed in the cytosol and, therefore, should not interfere with the biological activity of the oligonucleotides inside the cytosol of a cell. As a result, these larger chemical groups can be engineered to confer various advantages to the nucleotide or oligonucleotide, such as nuclease resistance, lipophilicity, charge, thermal stability, specificity, and reduced immunogenicity. In some embodiments, the structure of the glutathione-sensitive moiety can be engineered to modify the kinetics of its release.

In some embodiments, a glutathione-sensitive moiety is attached to the sugar of the nucleotide. In some embodiments, a glutathione-sensitive moiety is attached to the 2′-carbon of the sugar of a modified nucleotide. In some embodiments, the glutathione-sensitive moiety is located at the 5′-carbon of a sugar, particularly when the modified nucleotide is the 5′-terminal nucleotide of the oligonucleotide. In some embodiments, the glutathione-sensitive moiety is located at the 3′-carbon of a sugar, particularly when the modified nucleotide is the 3′-terminal nucleotide of the oligonucleotide. In some embodiments, the glutathione-sensitive moiety comprises a sulfonyl group. See, e.g., International Patent Application PCT/US2017/048239, which published on Mar. 1, 2018 as International Patent Publication WO2018/039364, entitled Compositions Comprising Reversibly Modified Oligonucleotides and Uses Thereof, which was filed on Aug. 23, 2016, the contents of which are incorporated by reference herein for its relevant disclosures.

v. Targeting Ligands

In some embodiments, it may be desirable to target the oligonucleotides of the disclosure to one or more cells or one or more organs. Such a strategy may help to avoid undesirable effects in other organs, or may avoid undue loss of the oligonucleotide to cells, tissue or organs that would not benefit for the oligonucleotide. Accordingly, in some embodiments, oligonucleotides disclosed herein may be modified to facilitate targeting of a particular tissue, cell or organ, e.g., to facilitate delivery of the oligonucleotide to the liver. In certain embodiments, oligonucleotides disclosed herein may be modified to facilitate delivery of the oligonucleotide to the hepatocytes of the liver. In some embodiments, an oligonucleotide comprises a nucleotide that is conjugated to one or more targeting ligands.

A targeting ligand may comprise a carbohydrate, amino sugar, cholesterol, peptide, polypeptide, protein or part of a protein (e.g., an antibody or antibody fragment) or lipid. In some embodiments, a targeting ligand is an aptamer. For example, a targeting ligand may be an RGD peptide that is used to target tumor vasculature or glioma cells, CREKA peptide to target tumor vasculature or stoma, transferrin, lactoferrin, or an aptamer to target transferrin receptors expressed on CNS vasculature, or an anti-EGFR antibody to target EGFR on glioma cells. In certain embodiments, the targeting ligand is one or more GalNAc moieties.

In some embodiments, 1 or more (e.g., 1, 2, 3, 4, 5, or 6) nucleotides of an oligonucleotide are each conjugated to a separate targeting ligand. In some embodiments, 2 to 4 nucleotides of an oligonucleotide are each conjugated to a separate targeting ligand. In some embodiments, targeting ligands are conjugated to 2 to 4 nucleotides at either ends of the sense or antisense strand (e.g., ligands are conjugated to a 2 to 4 nucleotide overhang or extension on the 5′ or 3′ end of the sense or antisense strand) such that the targeting ligands resemble bristles of a toothbrush and the oligonucleotide resembles a toothbrush. For example, an oligonucleotide may comprise a stem-loop at either the 5′ or 3′ end of the sense strand and 1, 2, 3, or 4 nucleotides of the loop of the stem may be individually conjugated to a targeting ligand, as described, for example, in International Patent Application Publication WO 2016/100401, which was published on Jun. 23, 2016, the relevant contents of which are incorporated herein by reference.

In some embodiments, it is desirable to target an oligonucleotide that reduces the expression of GYS2 to the hepatocytes of the liver of a subject. Any suitable hepatocyte targeting moiety may be used for this purpose.

GalNAc is a high affinity ligand for asialoglycoprotein receptor (ASGPR), which is primarily expressed on the sinusoidal surface of hepatocyte cells and has a major role in binding, internalization, and subsequent clearance of circulating glycoproteins that contain terminal galactose or N-acetylgalactosamine residues (asialoglycoproteins). Conjugation (either indirect or direct) of GalNAc moieties to oligonucleotides of the instant disclosure may be used to target these oligonucleotides to the ASGPR expressed on these hepatocyte cells.

In some embodiments, an oligonucleotide of the instant disclosure is conjugated directly or indirectly to a monovalent GalNAc. In some embodiments, the oligonucleotide is conjugated directly or indirectly to more than one monovalent GalNAc (i.e., is conjugated to 2, 3, or 4 monovalent GalNAc moieties, and is typically conjugated to 3 or 4 monovalent GalNAc moieties). In some embodiments, an oligonucleotide of the instant disclosure is conjugated to one or more bivalent GalNAc, trivalent GalNAc, or tetravalent GalNAc moieties.

In some embodiments, 1 or more (e.g., 1, 2, 3, 4, 5, or 6) nucleotides of an oligonucleotide are each conjugated to a GalNAc moiety. In some embodiments, 2 to 4 nucleotides of the loop (L) of the stem-loop are each conjugated to a separate GalNAc. In some embodiments, targeting ligands are conjugated to 2 to 4 nucleotides at either ends of the sense or antisense strand (e.g., ligands are conjugated to a 2 to 4 nucleotide overhang or extension on the 5′ or 3′ end of the sense or antisense strand) such that the GalNAc moieties resemble bristles of a toothbrush and the oligonucleotide resembles a toothbrush. For example, an oligonucleotide may comprise a stem-loop at either the 5′ or 3′ end of the sense strand and 1, 2, 3, or 4 nucleotides of the loop of the stem may be individually conjugated to a GalNAc moiety. In some embodiments, GalNAc moieties are conjugated to a nucleotide of the sense strand. For example, four GalNAc moieties can be conjugated to nucleotides in the tetraloop of the sense strand, where each GalNAc moiety is conjugated to one nucleotide.

Appropriate methods or chemistry (e.g., click chemistry) can be used to link a targeting ligand to a nucleotide. In some embodiments, a targeting ligand is conjugated to a nucleotide using a click linker. In some embodiments, an acetal-based linker is used to conjugate a targeting ligand to a nucleotide of any one of the oligonucleotides described herein. Acetal-based linkers are disclosed, for example, in International Patent Application Publication Number WO2016100401 A1, which published on Jun. 23, 2016, and the contents of which relating to such linkers are incorporated herein by reference. In some embodiments, the linker is a labile linker. However, in other embodiments, the linker is fairly stable. In some embodiments, a duplex extension (up to 3, 4, 5, or 6 base pairs in length) is provided between a targeting ligand (e.g., a GalNAc moiety) and a double-stranded oligonucleotide.

III. Formulations

Various formulations have been developed to facilitate oligonucleotide use. For example, oligonucleotides can be delivered to a subject or a cellular environment using a formulation that minimizes degradation, facilitates delivery and/or uptake, or provides another beneficial property to the oligonucleotides in the formulation. In some embodiments, provided herein are compositions comprising oligonucleotides (e.g., single-stranded or double-stranded oligonucleotides) to reduce the expression of GYS2. Such compositions can be suitably formulated such that when administered to a subject, either into the immediate environment of a target cell or systemically, a sufficient portion of the oligonucleotides enter the cell to reduce GYS2 expression. Any of a variety of suitable oligonucleotide formulations can be used to deliver oligonucleotides for the reduction of GYS2 as disclosed herein. In some embodiments, an oligonucleotide is formulated in buffer solutions such as phosphate-buffered saline solutions, liposomes, micellar structures, and capsids. In some embodiments, naked oligonucleotides or conjugates thereof are formulated in water or in an aqueous solution (e.g., water with pH adjustments). In some embodiments, naked oligonucleotides or conjugates thereof are formulated in basic buffered aqueous solutions (e.g., PBS)

Formulations of oligonucleotides with cationic lipids can be used to facilitate transfection of the oligonucleotides into cells. For example, cationic lipids, such as lipofectin, cationic glycerol derivatives, and polycationic molecules (e.g., polylysine) can be used. Suitable lipids include Oligofectamine, Lipofectamine (Life Technologies), NC388 (Ribozyme Pharmaceuticals, Inc., Boulder, Colo.), or FuGene 6 (Roche) all of which can be used according to the manufacturer's instructions.

Accordingly, in some embodiments, a formulation comprises a lipid nanoparticle. In some embodiments, an excipient comprises a liposome, a lipid, a lipid complex, a microsphere, a microparticle, a nanosphere, or a nanoparticle, or may be otherwise formulated for administration to the cells, tissues, organs, or body of a subject in need thereof (see, e.g., Remington: The Science and Practice of Pharmacy, 22nd edition, Pharmaceutical Press, 2013).

In some embodiments, formulations as disclosed herein comprise an excipient. In some embodiments, an excipient confers to a composition improved stability, improved absorption, improved solubility and/or therapeutic enhancement of the active ingredient. In some embodiments, an excipient is a buffering agent (e.g., sodium citrate, sodium phosphate, a tris base, or sodium hydroxide) or a vehicle (e.g., a buffered solution, petrolatum, dimethyl sulfoxide, or mineral oil). In some embodiments, an oligonucleotide is lyophilized for extending its shelf-life and then made into a solution before use (e.g., administration to a subject). Accordingly, an excipient in a composition comprising any one of the oligonucleotides described herein may be a lyoprotectant (e.g., mannitol, lactose, polyethylene glycol, or polyvinyl pyrolidone), or a collapse temperature modifier (e.g., dextran, ficoll, or gelatin).

In some embodiments, a pharmaceutical composition is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration. Typically, the route of administration is intravenous or subcutaneous.

Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous or subcutaneous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, and sodium chloride in the composition. Sterile injectable solutions can be prepared by incorporating the oligonucleotides in a required amount in a selected solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.

In some embodiments, a composition may contain at least about 0.1% of the therapeutic agent (e.g., an oligonucleotide for reducing GYS2 expression) or more, although the percentage of the active ingredient(s) may be between about 1% and about 80% or more of the weight or volume of the total composition. Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, as well as other pharmacological considerations will be contemplated by one skilled in the art of preparing such pharmaceutical formulations, and as such, a variety of dosages and treatment regimens may be desirable.

Even though a number of embodiments are directed to liver-targeted delivery of any of the oligonucleotides disclosed herein, targeting of other tissues is also contemplated.

IV. Methods of Use

i. Reducing GYS2 Expression in Cells

In some embodiments, methods are provided for delivering to a cell an effective amount any one of oligonucleotides disclosed herein for purposes of reducing expression of GYS2 in the cell. Methods provided herein are useful in any appropriate cell type. In some embodiments, a cell is any cell that expresses GYS2 (e.g., liver cells such as hepatocytes or adipose cells). In some embodiments, the cell is a primary cell that has been obtained from a subject and that may have undergone a limited number of a passages, such that the cell substantially maintains its natural phenotypic properties. In some embodiments, a cell to which the oligonucleotide is delivered is ex vivo or in vitro (i.e., can be delivered to a cell in culture or to an organism in which the cell resides). In specific embodiments, methods are provided for delivering to a cell an effective amount any one of the oligonucleotides disclosed herein for purposes of reducing expression of GYS2 solely or primarily in hepatocytes.

In some embodiments, oligonucleotides disclosed herein can be introduced using appropriate nucleic acid delivery methods including injection of a solution containing the oligonucleotides, bombardment by particles covered by the oligonucleotides, exposing the cell or organism to a solution containing the oligonucleotides, or electroporation of cell membranes in the presence of the oligonucleotides. Other appropriate methods for delivering oligonucleotides to cells may be used, such as lipid-mediated carrier transport, chemical-mediated transport, and cationic liposome transfection such as calcium phosphate, and others.

The consequences of inhibition can be confirmed by an appropriate assay to evaluate one or more properties of a cell or subject, or by biochemical techniques that evaluate molecules indicative of GYS2 expression (e.g., RNA, protein). In some embodiments, the extent to which an oligonucleotide provided herein reduces levels of expression of GYS2 is evaluated by comparing expression levels (e.g., mRNA or protein levels of GYS2 to an appropriate control (e.g., a level of GYS2 expression in a cell or population of cells to which an oligonucleotide has not been delivered or to which a negative control has been delivered). In some embodiments, an appropriate control level of GYS2 expression may be a predetermined level or value, such that a control level need not be measured every time. The predetermined level or value can take a variety of forms. In some embodiments, a predetermined level or value can be single cut-off value, such as a median or mean.

In some embodiments, administration of an oligonucleotide as described herein results in a reduction in the level of GYS2 expression in a cell. In some embodiments, the reduction in levels of GYS2 expression may be a reduction to 1% or lower, 5% or lower, 10% or lower, 15% or lower, 20% or lower, 25% or lower, 30% or lower, 35% or lower, 40% or lower, 45% or lower, 50% or lower, 55% or lower, 60% or lower, 70% or lower, 80% or lower, or 90% or lower compared with an appropriate control level of GYS2. The appropriate control level may be a level of GYS2 expression in a cell or population of cells that has not been contacted with an oligonucleotide as described herein. In some embodiments, the effect of delivery of an oligonucleotide to a cell according to a method disclosed herein is assessed after a finite period of time. For example, levels of GYS2 may be analyzed in a cell at least 8 hours, 12 hours, 18 hours, 24 hours; or at least one, two, three, four, five, six, seven, or fourteen days after introduction of the oligonucleotide into the cell.

In some embodiments, an oligonucleotide is delivered in the form of a transgene that is engineered to express in a cell the oligonucleotides (e.g., its sense and antisense strands). In some embodiments, an oligonucleotide is delivered using a transgene that is engineered to express any oligonucleotide disclosed herein. Transgenes may be delivered using viral vectors (e.g., adenovirus, retrovirus, vaccinia virus, poxvirus, adeno-associated virus or herpes simplex virus) or non-viral vectors (e.g., plasmids or synthetic mRNAs). In some embodiments, transgenes can be injected directly to a subject.

ii. Treatment Methods

Aspects of the disclosure relate to methods for reducing GYS2 expression for the treatment of a glycogen storage disease in a subject. In some embodiments, the methods may comprise administering to a subject in need thereof an effective amount of any one of the oligonucleotides disclosed herein. Such treatments could be used, for example, to decrease or prevent hepatomegaly, liver toxicity (e.g., lower or decrease levels of AST, ALT, and/or ALP), liver fibrosis, fatty acid deposition in the liver, hepatic hyperplasia, hepatocellular adenoma, and/or hepatocellular carcinoma. Such treatments could also be used, for example, to treat or prevent one or more symptoms associated with a glycogen storage disease selected from the list consisting of: GSDIa, GSDIII, GSDIV, GSDVI, and GSDIX, or to treat or prevent one or more symptoms of such a glycogen storage disease. The present disclosure provides for both prophylactic and therapeutic methods of treating a subject at risk of (or susceptible to) a glycogen storage disease (e.g., GSDIa, GSDIII, GSDIV, GSDVI, and GSDIX) and/or symptoms or conditions associated with a glycogen storage disease (e.g., GSDIa, GSDIII, GSDIV, GSDVI, and GSDIX).

In certain aspects, the disclosure provides a method for preventing in a subject, a disease, disorder, symptom, or condition as described herein by administering to the subject a therapeutic agent (e.g., an oligonucleotide or vector or transgene encoding same). In some embodiments, the subject to be treated is a subject who will benefit therapeutically from a reduction in the amount of GYS2 protein, e.g., in the liver.

Methods described herein typically involve administering to a subject an effective amount of an oligonucleotide, that is, an amount capable of producing a desirable therapeutic result. A therapeutically acceptable amount may be an amount that is capable of treating a disease or disorder. The appropriate dosage for any one subject will depend on certain factors, including the subject's size, body surface area, age, the particular composition to be administered, the active ingredient(s) in the composition, time and route of administration, general health, and other drugs being administered concurrently.

In some embodiments, a subject is administered any one of the compositions disclosed herein either enterally (e.g., orally, by gastric feeding tube, by duodenal feeding tube, via gastrostomy or rectally), parenterally (e.g., subcutaneous injection, intravenous injection or infusion, intra-arterial injection or infusion, intramuscular injection,), topically (e.g., epicutaneous, inhalational, via eye drops, or through a mucous membrane), or by direct injection into a target organ (e.g., the liver of a subject). Typically, oligonucleotides disclosed herein are administered intravenously or subcutaneously.

In some embodiments, oligonucleotides are administered at a dose in a range of 0.1 mg/kg to 25 mg/kg (e.g., 1 mg/kg to 5 mg/kg). In some embodiments, oligonucleotides are administered at a dose in a range of 0.1 mg/kg to 5 mg/kg or in a range of 0.5 mg/kg to 5 mg/kg.

As a non-limiting set of examples, the oligonucleotides of the instant disclosure would typically be administered once per year, twice per year, quarterly (once every three months), bi-monthly (once every two months), monthly, or weekly.

In some embodiments, the subject to be treated is a human (e.g., a human patient) or non-human primate or other mammalian subject. Other exemplary subjects include domesticated animals such as dogs and cats; livestock such as horses, cattle, pigs, sheep, goats, and chickens; and animals such as mice, rats, guinea pigs, and hamsters.

EXAMPLES Example 1: Development of GYS2 Oligonucleotide Inhibitors Using Human and Mouse Cell-Based Assays

Human and mouse-based assays were used to develop candidate oligonucleotides for inhibition of GYS2 expression. First, a computer-based algorithm was used to generate candidate oligonucleotide sequences (25-27-mer) for GYS2 inhibition. Cell-based assays and PCR assays were then employed for evaluation of candidate oligonucleotides for their ability to reduce GYS2 expression.

The computer-based algorithm provided oligonucleotides that were complementary to the human GYS2 mRNA (SEQ ID NO: 609, Table 1), of which certain sequences were also complementary to the Rhesus macaque GYS2 mRNA (SEQ ID NO: 610, Table 1).

TABLE 1 Sequences of human and Rhesus macaque GYS2 mRNA Species GenBank RefSeq # SEQ ID NO. Human NM_021957.3 609 Rhesus macaque XM_001098578.2 610

Of the oligonucleotides that the algorithm provided, 264 oligonucleotides were selected as candidates for experimental evaluation in a HEK-293 cell-based assay. In this assay, HEK-293 human embryonic kidney cells stably expressing GYS2 (referred to as HEK-GYS2 cells) were transfected with the oligonucleotides. Cells were maintained for a period of time following transfection and then levels of remaining GYS2 mRNA were interrogated using TAQMAN®-based qPCR assays. Two qPCR assays, a 3′ assay and a 5′ assay, were used to determine mRNA levels as measured by HEX and FAM probes, respectively. The results of the HEK-293 cell-based assay with the 264 oligonucleotides are shown in FIGS. 1A and 1B. The percent mRNA remaining is shown for each of the 3′ assay (circle shapes) and the 5′ assay (diamond shapes). Oligonucleotides with the lowest percentage of mRNA remaining compared to negative controls were considered hits. Oligonucleotides with low complementarity to the human genome were used as negative controls.

Based on the activity and locations of these oligonucleotides, hotspots on the human GYS2 mRNA were defined. A hotspot was identified as a stretch on the human GYS2 mRNA sequence associated with at least two oligonucleotides resulting in mRNA levels that were less than or equal to 35% in either assay compared with controls. Accordingly, the following hotspots within the human GYS2 mRNA sequence were identified: 579-618, 691-738, 1089-1125, 1175-1211, 1431-1486, 2341-2383, 2497-2543, 2660-2698, 2808-2851, and 3014-3050.

The sequences of the hotspots are outlined in Table 2.

TABLE 2 Sequences of Hotspots Hotspot Position In Human SEQ GYS2 ID mRNA Sequence NO. 579-618 GATAGAAGGAAGTCCTTATGTGGTACTTTTTGACATA 599 GGC 691-738 GACCGAGAAGCCAATGATATGCTGATATTTGGATCTT 600 TAACTGCCTGG 1089-1125 TCCAAACGGCTTGAATGTTAAGAAATTTTCAGCAGTG 601 1175-1211 TTGTTCGAGGTCATTTCTATGGICATCTCGACTTTGA 602 1431-1486 TGCACATTCTGTGAAGGAAAAGTTTGGAAAAAAACTC 603 TATGATGCATTATTAAGAG 2341-2383 AAGCTGCATGGTGAATATAAGAACTGAATTCTACATG 604 TGCTGC 2497-2543 GTGGAAGAAATTGAGTGAATGACAATTTTGTAATTTA 605 GGATAAGATC 2660-2698 TTTCTCTTACTCTGTTTATTTTTAAATGATCATCATA 606 AT 2808-2851 TAGCTAGGTTTTTACTGATTATTTTCATTTTTCACAT 607 GCATCAG 3014-3050 TCTTACTGTAACATTTTTCTATTGTTTAAATAGAAAG 608 Dose Response Analysis

Of the 264 oligonucleotides evaluated in the initial HEK-293 cell-based assay, 71 particularly active oligonucleotides were selected as hits based on their ability to knock down GYS2 levels and were subjected to a secondary screen.

In this secondary screen, the candidate oligonucleotides were tested using the same assay as in the primary screen, but at two or three different concentrations (1 nM, 0.1 nM and 0.03 nM) (FIGS. 2A and 2B). The target mRNA levels were generally normalized based on splicing factor, arginine/serine-rich 9 (SFRS9), a housekeeping gene that provides a stable expression reference across samples, to generate the percent mRNA shown in FIGS. 2A and 2B. The tested oligonucleotides in each of FIGS. 2A and 2B are shown compared to negative control sequences (NC1) and mock transfection. All 71 oligonucleotides had the same modification pattern, designated M1, which contains a combination of ribonucleotides, deoxyribonucleotides and 2′-O-methyl modified nucleotides. The sequences of the 71 oligonucleotides tested are provided in Table 3.

TABLE 3 Candidate oligonucleotide Sequences for HEK-293 Cell-Based Assay Sense Corresponding Antisense Hs Rm SEQ ID NO. SEQ ID NO. X X 1-3, 5, 6, 8, 9, 11-13, 193-195, 197, 198, 200, 201, 15-17, 19, 21-24, 52-54, 203-205, 207-209, 211, 213- 59, 62, 72, 73, 77, 82, 216, 244-246, 251, 254, 264, 84-86, 89, 93-95, 98, 265, 269, 274, 276-278, 281, 104, 106, 109, 111, 114, 285-287, 290, 296, 298, 301, 116-118, 129, 141-144, 158, 303, 306, 308-310, 321, 333- 160, 161, 177, 185, 186, 189, 336, 350, 352, 353, 369, 377, 192, 468, 469, 472, 473, 378, 381, 384, 519, 520, 523, 478-480, 482, 483, 487, 524, 529-531, 533, 534, 538, 488, 490-492, 498 539, 541-543, 549 Hs: human and Rm: Rhesus macaque; the sense and antisense SEQ ID NO. columns provide the sense strand and respective antisense strand, in relative order, that are hybridized to make each oligonucleotide. For example, sense strand of SEQ ID NO: 1 hybridizes with antisense strand of SEQ ID NO: 193. At this stage, 36 of the most potent sequences from the testing were selected for further analysis. The selected sequences were converted to nicked tetraloop structure formats (a 36-mer passenger strand with a 22-mer guide strand). See FIG. 3 for a generic tetraloop structure. These oligonucleotides were then tested as before, evaluating each oligonucleotide at three concentrations for its ability to reduce GYS2 mRNA expression in HepG2 cells.

FIG. 4 shows data for oligonucleotides made from different base sequences with nicked tetraloop structures, each adapted to one or two different modification patterns. The X-axis lists the 3′ end of the sense strand targeted by the oligonucleotide evaluated. The target mRNA levels were normalized as described above to generate the percent mRNA shown in FIG. 4 , and the tested oligonucleotides are shown compared to negative control sequences (NC1) and mock transfection.

Certain tetraloop-modified oligonucleotides were further tested in monkey hepatocyte cells using the same modification patterns for each compound (FIG. 5 ) at 0.1 μM, 0.3 μM, and 1.0 μM. The tested oligonucleotides in FIG. 5 are shown compared untransfected cells. Certain oligonucleotides were further tested using a full dose response curve in HEK-293 cells in order to determine the half maximal inhibitory concentration (IC₅₀) for each compound (see FIGS. 6A and 6B).

In Vivo Murine Screening

Data from the above in vitro experiments were assessed to identify tetraloops and modification patterns that would improve delivery properties while maintaining activity for reduction of GYS2 expression in the mouse hepatocytes. Based on this analysis, select oligonucleotides were then conjugated to GalNAc moieties. Four GalNAc moieties were conjugated to nucleotides in the tetraloop of the sense strand. Conjugation was performed using a click linker. The GalNAc used was as shown below:

A total of 65 potent GalNAc-conjugated GYS2 oligonucleotides from 18 different base sequences and having different modification patterns with nicked tetraloop structures were tested. Selected GYS2 oligonucleotide sequences were active against human and monkey mRNA sequences but not mouse Gys2. GYS2 oligonucleotides were subcutaneously administered to CD-1 mice transiently expressing human GYS2 mRNA by hydrodynamic injection of a human GYS2 expression plasmid at 0.5-5 mg/kg. Mice were euthanized on day 4 following administration. Liver samples were obtained and RNA was extracted to evaluate GYS2 mRNA levels by RT-qPCR. The percent GYS2 mRNA as compared to PBS control mRNA was determined based on these measurements.

From the 65 conjugates tested, the eight most potent base sequences were identified and each tested with the same modification pattern by subcutaneous injection at 0.5 mg/kg to CD-1 mice transiently expressing human GYS2 mRNA. Mice were euthanized on day 4 following administration. Liver samples were obtained and RNA was extracted to evaluate GYS2 mRNA levels by RT-qPCR. The percent GYS2 mRNA as compared to PBS control mRNA was determined based on these measurements and is shown in FIG. 7 .

Materials and Methods

Transfection

For the first screen, Lipofectamine RNAiMAX™ was used to complex the oligonucleotides for efficient transfection. Oligonucleotides, RNAiMAX and Opti-MEM incubated together at room temperature for 20 minutes and then 50 μL of this mix was added per well to plates prior to transfection. Media was aspirated from a flask of actively passaging cells and the cells were incubated at 37° C. in the presence of trypsin for 3-5 minutes. After cells no longer adhered to the flask, cell growth media (lacking penicillin and streptomycin) was added to neutralize the trypsin and to suspend the cells. A 10 μL aliquot was removed and counted with a hemocytometer to quantify the cells on a per milliliter basis. For cells, 10,000 or 25,000 cells/well were seeded per well in media (e.g., 100 μL of media). A diluted cell suspension was added to the 96-well transfection plates, which already contained the oligonucleotides in Opti-MEM. The transfection plates were then incubated for 24 hours at 37° C. After 24 hours of incubation, media was aspirated from each well. Cells were lysed using the lysis buffer from the Promega RNA Isolation kit. The lysis buffer was added to each well. The lysed cells were then transferred to the Corbett XtractorGENE (QIAxtractor) for RNA isolation or stored at −80° C.

For subsequent screens and experiments, e.g., the secondary screen, Lipofectamine RNAiMAx was used to complex the oligonucleotides for reverse transfection. The complexes were made by mixing RNAiMAX and siRNAs in OptiMEM medium for 15 minutes. The transfection mixture was transferred to multi-well plates and cell suspension was added to the wells. After 24 hours incubation the cells were washed once with PBS and then lysed using lysis buffer from the Promega SV96 kit. The RNA was purified using the SV96 plates in a vacuum manifold. Four microliters of the purified RNA was then heated at 65° C. for 5 minutes and cooled to 4° C. The RNA was then used for reverse transcription using the High Capacity Reverse Transcription kit (Life Technologies) in a 10 microliter reaction. The cDNA was then diluted to 50 μL with nuclease free water and used for quantitative PCR with multiplexed 5′-endonuclease assays and SSoFast qPCR mastermix (Bio-Rad laboratories).

cDNA Synthesis

RNA was isolated from mammalian cells in tissue culture using the Corbett X-tractor Gene™ (QIAxtractor). A modified SuperScript II protocol was used to synthesize cDNA from the isolated RNA. Isolated RNA (approximately 5 ng/μL) was heated to 65° C. for five minutes and incubated with dNPs, random hexamers, oligo dTs, and water. The mixture was cooled for 15 seconds. An “enzyme mix,” consisting of water, 5× first strand buffer, DTT, SUPERase⋅In™ (an RNA inhibitor), and SuperScript II RTase was added to the mixture. The contents were heated to 42° C. for one hour, then to 70° C. for 15 minutes, and then cooled to 4° C. using a thermocycler. The resulting cDNA was then subjected to SYBR®-based qPCR. The qPCR reactions were multiplexed, containing two 5′ endonuclease assays per reaction.

qPCR Assays

Primer sets were initially screened using SYBR®-based qPCR. Assay specificity was verified by assessing melt curves as well as “minus RT” controls. Dilutions of cDNA template (10-fold serial dilutions from 20 ng and to 0.02 ng per reaction) from HeLa and Hepa1-6 cells were used to test human (Hs) and mouse (Mm) assays, respectively. qPCR assays were set up in 384-well plates, covered with MicroAmp film, and run on the 7900HT from Applied Biosystems. Reagent concentrations and cycling conditions included the following: 2×SYBR mix, 10 μM forward primer, 10 μM reverse primer, DD H₂O, and cDNA template up to a total volume of 10 μL.

Cloning

PCR amplicons that displayed a single melt-curve were ligated into the pGEM®-T Easy vector kit from Promega according to the manufacturer's instructions. Following the manufacturer's protocol, JM109 High Efficiency cells were transformed with the newly ligated vectors. The cells were then plated on LB plates containing ampicillin and incubated at 37° C. overnight for colony growth.

PCR Screening and Plasmid Mini-Prep

PCR was used to identify colonies of E. coli that had been transformed with a vector containing the ligated amplicon of interest. Vector-specific primers that flank the insert were used in the PCR reaction. All PCR products were then run on a 1% agarose gel and imaged by a transilluminator following staining. Gels were assessed qualitatively to determine which plasmids appeared to contain a ligated amplicon of the expected size (approximately 300 bp, including the amplicon and the flanking vector sequences specific to the primers used).

The colonies that were confirmed transformants by PCR screening were then incubated overnight in cultures consisting of 2 mL LB broth with ampicillin at 37° C. with shaking. E. coli cells were then lysed, and the plasmids of interest were isolated using Promega's Mini-Prep kit. Plasmid concentration was determined by UV absorbance at 260 nm.

Plasmid Sequencing and Quantification

Purified plasmids were sequenced using the BigDye® Terminator sequencing kit. The vector-specific primer, T7, was used to give read lengths that span the insert. The following reagents were used in the sequencing reactions: water, 5× sequencing buffer, BigDye terminator mix, T7 primer, and plasmid (100 ng/μL) to a volume of 10 μL. The mixture was held at 96° C. for one minute, then subjected to 15 cycles of 96° C. for 10 seconds, 50° C. for 5 seconds, 60° C. for 1 minute, 15 seconds; 5 cycles of 96° C. for 10 seconds, 50° C. for 5 seconds, 60° C. for 1 minute, 30 seconds; and 5 cycles of 96° C. for 10 seconds, 50° C. for 5 seconds, and 60° C. for 2 minutes. Dye termination reactions were then sequenced using Applied Biosystems' capillary electrophoresis sequencers.

Sequence-verified plasmids were then quantified. They were linearized using a single cutting restriction endonuclease. Linearity was confirmed using agarose gel electrophoresis. All plasmid dilutions were made in TE buffer (pH 7.5) with 100 μg of tRNA per mL buffer to reduce non-specific binding of plasmid to the polypropylene vials.

The linearized plasmids were then serially diluted from 1,000,000 to 01 copies per μL and subjected to qPCR. Assay efficiency was calculated and the assays were deemed acceptable if the efficiency was in the range of 90-110%.

Multi-Plexing Assays

For each target, mRNA levels were quantified by two 5′ nuclease assays. In general, several assays are screened for each target. The two assays selected displayed a combination of good efficiency, low limit of detection, and broad 5′→3′ coverage of the gene of interest (GOI). Both assays against one GOI could be combined in one reaction when different fluorophores were used on the respective probes. Thus, the final step in assay validation was to determine the efficiency of the selected assays when they were combined in the same qPCR or “multi-plexed.”

Linearized plasmids for both assays in 10-fold dilutions were combined and qPCR was performed. The efficiency of each assay was determined as described above. The accepted efficiency rate was 90-110%. While validating multi-plexed reactions using linearized plasmid standards, C_(q) values for the target of interest were also assessed using cDNA as the template. For human or mouse targets, HeLa and Hepa1-6 cDNA were used, respectively. The cDNA, in this case, was derived from RNA isolated on the Corbett (˜5 ng/μl in water) from untransfected cells. In this way, the observed C_(q) values from this sample cDNA were representative of the expected C_(q) values from a 96-well plate transfection. In cases where C_(q) values were greater than 30, other cell lines were sought that exhibit higher expression levels of the gene of interest. A library of total RNA isolated from via high-throughput methods on the Corbett from each human and mouse line was generated and used to screen for acceptable levels of target expression.

Description of Oligonucleotide Nomenclature

All oligonucleotides described herein are designated either SN₁-ASN₂-MN₃. The following designations apply:

-   -   N₁: sequence identifier number of the sense strand sequence     -   N₂: sequence identifier number of the antisense strand sequence     -   N₃: reference number of modification pattern, in which each         number represents a pattern of modified nucleotides in the         oligonucleotide.         For example, S27-AS219-M1 represents an oligonucleotide with a         sense sequence that is set forth by SEQ ID NO: 27, an antisense         sequence that is set forth by SEQ ID NO: 219, and which is         adapted to a modification pattern identified as M1.

TABLE 4 GYS2 RNAi oligonucleotides S SEQ AS SEQ App Name Sense Sequence ID NO Antisense Sequence ID NO S1-AS193- CAGGUGCAUUUUGGAAGAUGGCUGA   1 UCAGCCAUCUUCCAAAAUG 193 M1 CACCUGGC S2-AS194- AGGUGCAUUUUGGAAGAUGGCUGAT   2 AUCAGCCAUCUUCCAAAAU 194 M1 GCACCUGG S3-AS195- GGUGCAUUUUGGAAGAUGGCUGATA   3 UAUCAGCCAUCUUCCAAAA 195 M1 UGCACCUG S4-AS196- GUGCAUUUUGGAAGAUGGCUGAUAG   4 CUAUCAGCCAUCUUCCAAA 196 M1 AUGCACCU S5-AS197- UGCAUUUUGGAAGAUGGCUGAUAGA   5 UCUAUCAGCCAUCUUCCAA 197 M1 AAUGCACC S6-AS198- AAGGAAGUCCUUAUGUGGUACUUTT   6 AAAAGUACCACAUAAGGAC 198 M1 UUCCUUCU S7-AS199- GAAGUCCUUAUGUGGUACUUUUUGA   7 UCAAAAAGUACCACAUAAG 199 M1 GACUUCCU S8-AS200- AAGUCCUUAUGUGGUACUUUUUGAC   8 GUCAAAAAGUACCACAUAA 200 M1 GGACUUCC S9-AS201- AGUCCUUAUGUGGUACUUUUUGACA   9 UGUCAAAAAGUACCACAUA 201 M1 AGGACUUC S10-AS202- GUCCUUAUGUGGUACUUUUUGACAT  10 AUGUCAAAAAGUACCACAU 202 M1 AAGGACUU S11-AS203- CCUUAUGUGGUACUUUUUGACAUAG  11 CUAUGUCAAAAAGUACCAC 203 M1 AUAAGGAC S12-AS204- GUACUUUUUGACAUAGGCUAUUCAG  12 CUGAAUAGCCUAUGUCAAA 204 M1 AAGUACCA S13-AS205- CGAGAAGCCAAUGAUAUGCUGAUAT  13 AUAUCAGCAUAUCAUUGG 205 M1 CUUCUCGGU S14-AS206- GAGAAGCCAAUGAUAUGCUGAUATT  14 AAUAUCAGCAUAUCAUUG 206 M1 GCUUCUCGG S15-AS207- AGAAGCCAAUGAUAUGCUGAUAUTT  15 AAAUAUCAGCAUAUCAUU 207 M1 GGCUUCUCG S16-AS208- GAAGCCAAUGAUAUGCUGAUAUUTG  16 CAAAUAUCAGCAUAUCAUU 208 M1 GGCUUCUC S17-AS209- AGCCAAUGAUAUGCUGAUAUUUGGA  17 UCCAAAUAUCAGCAUAUCA 209 M1 UUGGCUUC S18-AS210- GCCAAUGAUAUGCUGAUAUUUGGAT  18 AUCCAAAUAUCAGCAUAUC 210 M1 AUUGGCUU S19-AS211- CCAAUGAUAUGCUGAUAUUUGGATC  19 GAUCCAAAUAUCAGCAUAU 211 M1 CAUUGGCU S20-AS212- AAUGAUAUGCUGAUAUUUGGAUCTT  20 AAGAUCCAAAUAUCAGCAU 212 M1 AUCAUUGG S21-AS213- AUGAUAUGCUGAUAUUUGGAUCUTT  21 AAAGAUCCAAAUAUCAGCA 213 M1 UAUCAUUG S22-AS214- UGAUAUGCUGAUAUUUGGAUCUUTA  22 UAAAGAUCCAAAUAUCAGC 214 M1 AUAUCAUU S23-AS215- AUAUGCUGAUAUUUGGAUCUUUAAC  23 GUUAAAGAUCCAAAUAUCA 215 M1 GCAUAUCA S24-AS216- AUGCUGAUAUUUGGAUCUUUAACTG  24 CAGUUAAAGAUCCAAAUAU 216 M1 CAGCAUAU S25-AS217- UCUUUAACUGCCUGGUUCUUAAAAG  25 CUUUUAAGAACCAGGCAG 217 M1 UUAAAGAUC S26-AS218- CUUUAACUGCCUGGUUCUUAAAAGA  26 UCUUUUAAGAACCAGGCA 218 M1 GUUAAAGAU S27-AS219- UUUAACUGCCUGGUUCUUAAAAGAG  27 CUCUUUUAAGAACCAGGCA 219 M1 GUUAAAGA S28-AS220- UUAACUGCCUGGUUCUUAAAAGAGG  28 CCUCUUUUAAGAACCAGGC 220 M1 AGUUAAAG S29-AS221- UAACUGCCUGGUUCUUAAAAGAGGT  29 ACCUCUUUUAAGAACCAGG 221 M1 CAGUUAAA S30-AS222- UUGCCCAAUUCCAUGAAUGGCAGGC  30 GCCUGCCAUUCAUGGAAU 222 M1 UGGGCAACG S31-AS223- UGCCCAAUUCCAUGAAUGGCAGGCT  31 AGCCUGCCAUUCAUGGAAU 223 M1 UGGGCAAC S32-AS224- GCCCAAUUCCAUGAAUGGCAGGCTG  32 CAGCCUGCCAUUCAUGGAA 224 M1 UUGGGCAA S33-AS225- CCCAAUUCCAUGAAUGGCAGGCUGG  33 CCAGCCUGCCAUUCAUGGA 225 M1 AUUGGGCA S34-AS226- CCAAUUCCAUGAAUGGCAGGCUGGA  34 UCCAGCCUGCCAUUCAUGG 226 M1 AAUUGGGC S35-AS227- CAAUUCCAUGAAUGGCAGGCUGGAA  35 UUCCAGCCUGCCAUUCAUG 227 M1 GAAUUGGG S36-AS228- AAUUCCAUGAAUGGCAGGCUGGAAT  36 AUUCCAGCCUGCCAUUCAU 228 M1 GGAAUUGG S37-AS229- AUUCCAUGAAUGGCAGGCUGGAATT  37 AAUUCCAGCCUGCCAUUCA 229 M1 UGGAAUUG S38-AS230- UUCCAUGAAUGGCAGGCUGGAAUTG  38 CAAUUCCAGCCUGCCAUUC 230 M1 AUGGAAUU S39-AS231- UCCAUGAAUGGCAGGCUGGAAUUGG  39 CCAAUUCCAGCCUGCCAUU 231 M1 CAUGGAAU S40-AS232- GGAAACUUCCUAUUGCCACAAUATT  40 AAUAUUGUGGCAAUAGGA 232 M1 AGUUUCCUG S41-AS233- GGUAUCUCUGUGCAGCAAAUAUUGA  41 UCAAUAUUUGCUGCACAG 233 M1 AGAUACCUC S42-AS234- GUAUCUCUGUGCAGCAAAUAUUGAT  42 AUCAAUAUUUGCUGCACA 234 M1 GAGAUACCU S43-AS235- UAUCUCUGUGCAGCAAAUAUUGATT  43 AAUCAAUAUUUGCUGCACA 235 M1 GAGAUACC S44-AS236- AUCUCUGUGCAGCAAAUAUUGAUTT  44 AAAUCAAUAUUUGCUGCAC 236 M1 AGAGAUAC S45-AS237- UCUCUGUGCAGCAAAUAUUGAUUTC  45 GAAAUCAAUAUUUGCUGC 237 M1 ACAGAGAUA S46-AS238- CUCUGUGCAGCAAAUAUUGAUUUCT  46 AGAAAUCAAUAUUUGCUG 238 M1 CACAGAGAU S47-AS239- UCUGUGCAGCAAAUAUUGAUUUCTA  47 UAGAAAUCAAUAUUUGCU 239 M1 GCACAGAGA S48-AS240- CUGUGCAGCAAAUAUUGAUUUCUAC  48 GUAGAAAUCAAUAUUUGC 240 M1 UGCACAGAG S49-AS241- UGUGCAGCAAAUAUUGAUUUCUACA  49 UGUAGAAAUCAAUAUUUG 241 M1 CUGCACAGA S50-AS242- GUGCAGCAAAUAUUGAUUUCUACAA  50 UUGUAGAAAUCAAUAUUU 242 M1 GCUGCACAG S51-AS243- UGCAGCAAAUAUUGAUUUCUACAAC  51 GUUGUAGAAAUCAAUAUU 243 M1 UGCUGCACA S52-AS244- GCAGCAAAUAUUGAUUUCUACAACC  52 GGUUGUAGAAAUCAAUAU 244 M1 UUGCUGCAC S53-AS245- CAGCAAAUAUUGAUUUCUACAACCA  53 UGGUUGUAGAAAUCAAUA 245 M1 UUUGCUGCA S54-AS246- AGCAAAUAUUGAUUUCUACAACCAT  54 AUGGUUGUAGAAAUCAAU 246 M1 AUUUGCUGC S55-AS247- GCAAAUAUUGAUUUCUACAACCATC  55 GAUGGUUGUAGAAAUCAA 247 M1 UAUUUGCUG S56-AS248- AUAUUGAUUUCUACAACCAUCUUGA  56 UCAAGAUGGUUGUAGAAA 248 M1 UCAAUAUUU S57-AS249- UUGAUUUCUACAACCAUCUUGAUAA  57 UUAUCAAGAUGGUUGUAG 249 M1 AAAUCAAUA S58-AS250- GAUUUCUACAACCAUCUUGAUAAGT  58 ACUUAUCAAGAUGGUUGU 250 M1 AGAAAUCAA S59-AS251- AUUUCUACAACCAUCUUGAUAAGTT  59 AACUUAUCAAGAUGGUUG 251 M1 UAGAAAUCA S60-AS252- UUCUACAACCAUCUUGAUAAGUUTA  60 UAAACUUAUCAAGAUGGU 252 M1 UGUAGAAAU S61-AS253- CUACAACCAUCUUGAUAAGUUUAAC  61 GUUAAACUUAUCAAGAUG 253 M1 GUUGUAGAA S62-AS254- UACAACCAUCUUGAUAAGUUUAACA  62 UGUUAAACUUAUCAAGAU 254 M1 GGUUGUAGA S63-AS255- ACAACCAUCUUGAUAAGUUUAACAT  63 AUGUUAAACUUAUCAAGA 255 M1 UGGUUGUAG S64-AS256- CAACCAUCUUGAUAAGUUUAACATT  64 AAUGUUAAACUUAUCAAG 256 M1 AUGGUUGUA S65-AS257- AACCAUCUUGAUAAGUUUAACAUTG  65 CAAUGUUAAACUUAUCAA 257 M1 GAUGGUUGU S66-AS258- ACCAUCUUGAUAAGUUUAACAUUGA  66 UCAAUGUUAAACUUAUCA 258 M1 AGAUGGUUG S67-AS259- CCAUCUUGAUAAGUUUAACAUUGAC  67 GUCAAUGUUAAACUUAUC 259 M1 AAGAUGGUU S68-AS260- CAUCUUGAUAAGUUUAACAUUGACA  68 UGUCAAUGUUAAACUUAU 260 M1 CAAGAUGGU S69-AS261- AUCUUGAUAAGUUUAACAUUGACAA  69 UUGUCAAUGUUAAACUUA 261 M1 UCAAGAUGG S70-AS262- GUUCACCACGGUUUCUGAAAUAACA  70 UGUUAUUUCAGAAACCGU 262 M1 GGUGAACAC S71-AS263- CACCACGGUUUCUGAAAUAACAGCA  71 UGCUGUUAUUUCAGAAAC 263 M1 CGUGGUGAA S72-AS264- CCACGGUUUCUGAAAUAACAGCAAT  72 AUUGCUGUUAUUUCAGAA 264 M1 ACCGUGGUG S73-AS265- CACGGUUUCUGAAAUAACAGCAATA  73 UAUUGCUGUUAUUUCAGA 265 M1 AACCGUGGU S74-AS266- CGGUUUCUGAAAUAACAGCAAUAGA  74 UCUAUUGCUGUUAUUUCA 266 M1 GAAACCGUG S75-AS267- GGUUUCUGAAAUAACAGCAAUAGAA  75 UUCUAUUGCUGUUAUUUC 267 M1 AGAAACCGU S76-AS268- GUUUCUGAAAUAACAGCAAUAGAAG  76 CUUCUAUUGCUGUUAUUU 268 M1 CAGAAACCG S77-AS269- CUGAAAUAACAGCAAUAGAAGCUGA  77 UCAGCUUCUAUUGCUGUU 269 M1 AUUUCAGAA S78-AS270- AAGAGAAAGCCUGAUGUAGUUACTC  78 GAGUAACUACAUCAGGCU 270 M1 UUCUCUUCA S79-AS271- AGAGAAAGCCUGAUGUAGUUACUCC  79 GGAGUAACUACAUCAGGC 271 M1 UUUCUCUUC S80-AS272- GGCUUGAAUGUUAAGAAAUUUUCAG  80 CUGAAAAUUUCUUAACAU 272 M1 UCAAGCCGU S81-AS273- GCUUGAAUGUUAAGAAAUUUUCAGC  81 GCUGAAAAUUUCUUAACA 273 M1 UUCAAGCCG S82-AS274- CUUGAAUGUUAAGAAAUUUUCAGCA  82 UGCUGAAAAUUUCUUAAC 274 M1 AUUCAAGCC S83-AS275- UUGAAUGUUAAGAAAUUUUCAGCAG  83 CUGCUGAAAAUUUCUUAA 275 M1 CAUUCAAGC S84-AS276- UGAAUGUUAAGAAAUUUUCAGCAGT  84 ACUGCUGAAAAUUUCUUA 276 M1 ACAUUCAAG S85-AS277- GAAUGUUAAGAAAUUUUCAGCAGTG  85 CACUGCUGAAAAUUUCUU 277 M1 AACAUUCAA S86-AS278- AUGUUAAGAAAUUUUCAGCAGUGCA  86 UGCACUGCUGAAAAUUUC 278 M1 UUAACAUUC S87-AS279- AGAAAUUUUCAGCAGUGCAUGAGTT  87 AACUCAUGCACUGCUGAAA 279 M1 AUUUCUUA S88-AS280- AGCAGUGCAUGAGUUUCAAAAUCTA  88 UAGAUUUUGAAACUCAUG 280 M1 CACUGCUGA S89-AS281- AGAUUUUGUUCGAGGUCAUUUCUAT  89 AUAGAAAUGACCUCGAACA 281 M1 AAAUCUUG S90-AS282- GUUCGAGGUCAUUUCUAUGGUCATC  90 GAUGACCAUAGAAAUGACC 282 M1 UCGAACAA S91-AS283- UUCGAGGUCAUUUCUAUGGUCAUCT  91 AGAUGACCAUAGAAAUGAC 283 M1 CUCGAACA S92-AS284- UCGAGGUCAUUUCUAUGGUCAUCTC  92 GAGAUGACCAUAGAAAUG 284 M1 ACCUCGAAC S93-AS285- CGAGGUCAUUUCUAUGGUCAUCUCG  93 CGAGAUGACCAUAGAAAUG 285 M1 ACCUCGAA S94-AS286- GAGGUCAUUUCUAUGGUCAUCUCGA  94 UCGAGAUGACCAUAGAAA 286 M1 UGACCUCGA S95-AS287- AGGUCAUUUCUAUGGUCAUCUCGAC  95 GUCGAGAUGACCAUAGAAA 287 M1 UGACCUCG S96-AS288- GGUCAUUUCUAUGGUCAUCUCGACT  96 AGUCGAGAUGACCAUAGAA 288 M1 AUGACCUC S97-AS289- GUCAUUUCUAUGGUCAUCUCGACTT  97 AAGUCGAGAUGACCAUAGA 289 M1 AAUGACCU S98-AS290- UGAAAAGACUUUGUUCCUUUUCATT  98 AAUGAAAAGGAACAAAGUC 290 M1 UUUUCAAG S99-AS291- GAAAAGACUUUGUUCCUUUUCAUTG  99 CAAUGAAAAGGAACAAAGU 291 M1 CUUUUCAA S100- AAAGACUUUGUUCCUUUUCAUUGCT 100 AGCAAUGAAAAGGAACAAA 292 AS292-M1 GUCUUUUC S101- CUGAGGAUGCAUAAAAGUGACAUCA 101 UGAUGUCACUUUUAUGCA 293 AS293-M1 UCCUCAGCA S102- GAGGAUGCAUAAAAGUGACAUCACA 102 UGUGAUGUCACUUUUAUG 294 AS294-M1 CAUCCUCAG S103- UUUUUCAUUAUGCCUGCCAAGACAA 103 UUGUCUUGGCAGGCAUAA 295 AS295-M1 UGAAAAACA S104- UCAUUAUGCCUGCCAAGACAAAUAA 104 UUAUUUGUCUUGGCAGGC 296 AS296-M1 AUAAUGAAA S105- CAUUAUGCCUGCCAAGACAAAUAAT 105 AUUAUUUGUCUUGGCAGG 297 AS297-M1 CAUAAUGAA S106- AUUAUGCCUGCCAAGACAAAUAATT 106 AAUUAUUUGUCUUGGCAG 298 AS298-M1 GCAUAAUGA S107- AUGCCUGCCAAGACAAAUAAUUUCA 107 UGAAAUUAUUUGUCUUGG 299 AS299-M1 CAGGCAUAA S108- AAUUUCAACGUGGAAACCCUGAAAG 108 CUUUCAGGGUUUCCACGU 300 AS300-M1 UGAAAUUAU S109- AUUUCAACGUGGAAACCCUGAAAGG 109 CCUUUCAGGGUUUCCACG 301 AS301-M1 UUGAAAUUA S110- UUUCAACGUGGAAACCCUGAAAGGA 110 UCCUUUCAGGGUUUCCAC 302 AS302-M1 GUUGAAAUU S111- UUCAACGUGGAAACCCUGAAAGGAC 111 GUCCUUUCAGGGUUUCCA 303 AS303-M1 CGUUGAAAU S112- UCAACGUGGAAACCCUGAAAGGACA 112 UGUCCUUUCAGGGUUUCC 304 AS304-M1 ACGUUGAAA S113- UUGCACAUUCUGUGAAGGAAAAGTT 113 AACUUUUCCUUCACAGAAU 305 AS305-M1 GUGCAACA S114- UGCACAUUCUGUGAAGGAAAAGUTT 114 AAACUUUUCCUUCACAGAA 306 AS306-M1 UGUGCAAC S115- GCACAUUCUGUGAAGGAAAAGUUTG 115 CAAACUUUUCCUUCACAGA 307 AS307-M1 AUGUGCAA S116- AUUCUGUGAAGGAAAAGUUUGGAAA 116 UUUCCAAACUUUUCCUUCA 308 AS308-M1 CAGAAUGU S117- GUGAAGGAAAAGUUUGGAAAAAAAC 117 GUUUUUUUCCAAACUUUU 309 AS309-M1 CCUUCACAG S118- GAAAAAAACUCUAUGAUGCAUUATT 118 AAUAAUGCAUCAUAGAGU 310 AS310-M1 UUUUUUCCA S119- AAAAAAACUCUAUGAUGCAUUAUTA 119 UAAUAAUGCAUCAUAGAG 311 AS311-M1 UUUUUUUCC S120- AAAAAACUCUAUGAUGCAUUAUUAA 120 UUAAUAAUGCAUCAUAGA 312 AS312-M1 GUUUUUUUC S121- AAAAACUCUAUGAUGCAUUAUUAAG 121 CUUAAUAAUGCAUCAUAG 313 AS313-M1 AGUUUUUUU S122- UUAUUAAGAGGAGAAAUUCCUGACC 122 GGUCAGGAAUUUCUCCUC 314 AS314-M1 UUAAUAAUG S123- UAUUAAGAGGAGAAAUUCCUGACCT 123 AGGUCAGGAAUUUCUCCU 315 AS315-M1 CUUAAUAAU S124- AUUAAGAGGAGAAAUUCCUGACCTG 124 CAGGUCAGGAAUUUCUCC 316 AS316-M1 UCUUAAUAA S125- UUAAGAGGAGAAAUUCCUGACCUGA 125 UCAGGUCAGGAAUUUCUC 317 AS317-M1 CUCUUAAUA S126- UAAGAGGAGAAAUUCCUGACCUGAA 126 UUCAGGUCAGGAAUUUCU 318 AS318-M1 CCUCUUAAU S127- AAGAGGAGAAAUUCCUGACCUGAAC 127 GUUCAGGUCAGGAAUUUC 319 AS319-M1 UCCUCUUAA S128- CGAGAUGAUCUAACAAUUAUGAAAA 128 UUUUCAUAAUUGUUAGAU 320 AS320-M1 CAUCUCGAU S129- AGAUGAUCUAACAAUUAUGAAAAGA 129 UCUUUUCAUAAUUGUUAG 321 AS321-M1 AUCAUCUCG S130- GAUGAUCUAACAAUUAUGAAAAGAG 130 CUCUUUUCAUAAUUGUUA 322 AS322-M1 GAUCAUCUC S131- AUGAUCUAACAAUUAUGAAAAGAGC 131 GCUCUUUUCAUAAUUGUU 323 AS323-M1 AGAUCAUCU S132- ACAAUUAUGAAAAGAGCCAUCUUTT 132 AAAAGAUGGCUCUUUUCA 324 AS324-M1 UAAUUGUUA S133- GAAAAGAGCCAUCUUUUCAACUCAG 133 CUGAGUUGAAAAGAUGGC 325 AS325-M1 UCUUUUCAU S134- CCCAUCCUCAGCACCAUUAGACGGA 134 UCCGUCUAAUGGUGCUGA 326 AS326-M1 GGAUGGGGU S135- CCAUCCUCAGCACCAUUAGACGGAT 135 AUCCGUCUAAUGGUGCUG 327 AS327-M1 AGGAUGGGG S136- CAUCCUCAGCACCAUUAGACGGATT 136 AAUCCGUCUAAUGGUGCU 328 AS328-M1 GAGGAUGGG S137- AUCCUCAGCACCAUUAGACGGAUTG 137 CAAUCCGUCUAAUGGUGC 329 AS329-M1 UGAGGAUGG S138- UCCUCAGCACCAUUAGACGGAUUGG 138 CCAAUCCGUCUAAUGGUGC 330 AS330-M1 UGAGGAUG S139- CCCAUGGACUAUGAAGAGUUUGUTA 139 UAACAAACUCUUCAUAGUC 331 AS331-M1 CAUGGGUA S140- CCAUGGACUAUGAAGAGUUUGUUAG 140 CUAACAAACUCUUCAUAGU 332 AS332-M1 CCAUGGGU S141- CUUGGAGUAUUUCCAUCAUACUATG 141 CAUAGUAUGAUGGAAAUA 333 AS333-M1 CUCCAAGAU S142- UGGAGUAUUUCCAUCAUACUAUGAA 142 UUCAUAGUAUGAUGGAAA 334 AS334-M1 UACUCCAAG S143- GGAGUAUUUCCAUCAUACUAUGAAC 143 GUUCAUAGUAUGAUGGAA 335 AS335-M1 AUACUCCAA S144- GAGUAUUUCCAUCAUACUAUGAACC 144 GGUUCAUAGUAUGAUGGA 336 AS336-M1 AAUACUCCA S145- UAUUUCCAUCAUACUAUGAACCCTG 145 CAGGGUUCAUAGUAUGAU 337 AS337-M1 GGAAAUACU S146- AUACUCCAGCUGAAUGCACUGUGAT 146 AUCACAGUGCAUUCAGCUG 338 AS338-M1 GAGUAUAA S147- GGCAGAUAUUACCAGCAUGCCAGAC 147 GUCUGGCAUGCUGGUAAU 339 AS339-M1 AUCUGCCUA S148- GCAGAUAUUACCAGCAUGCCAGACA 148 UGUCUGGCAUGCUGGUAA 340 AS340-M1 UAUCUGCCU S149- CAGAUAUUACCAGCAUGCCAGACAC 149 GUGUCUGGCAUGCUGGUA 341 AS341-M1 AUAUCUGCC S150- AGAUAUUACCAGCAUGCCAGACACC 150 GGUGUCUGGCAUGCUGGU 342 AS342-M1 AAUAUCUGC S151- GAUAUUACCAGCAUGCCAGACACCT 151 AGGUGUCUGGCAUGCUGG 343 AS343-M1 UAAUAUCUG S152- AUAUUACCAGCAUGCCAGACACCTG 152 CAGGUGUCUGGCAUGCUG 344 AS344-M1 GUAAUAUCU S153- UAUUACCAGCAUGCCAGACACCUGA 153 UCAGGUGUCUGGCAUGCU 345 AS345-M1 GGUAAUAUC S154- AUUACCAGCAUGCCAGACACCUGAC 154 GUCAGGUGUCUGGCAUGC 346 AS346-M1 UGGUAAUAU S155- UUACCAGCAUGCCAGACACCUGACA 155 UGUCAGGUGUCUGGCAUG 347 AS347-M1 CUGGUAAUA S156- UACCAGCAUGCCAGACACCUGACAT 156 AUGUCAGGUGUCUGGCAU 348 AS348-M1 GCUGGUAAU S157- ACCAGCAUGCCAGACACCUGACATT 157 AAUGUCAGGUGUCUGGCA 349 AS349-M1 UGCUGGUAA S158- CCAGCAUGCCAGACACCUGACAUTA 158 UAAUGUCAGGUGUCUGGC 350 AS350-M1 AUGCUGGUA S159- CAGCAUGCCAGACACCUGACAUUAA 159 UUAAUGUCAGGUGUCUGG 351 AS351-M1 CAUGCUGGU S160- AGCAUGCCAGACACCUGACAUUAAG 160 CUUAAUGUCAGGUGUCUG 352 AS352-M1 GCAUGCUGG S161- GCAUGCCAGACACCUGACAUUAAGC 161 GCUUAAUGUCAGGUGUCU 353 AS353-M1 GGCAUGCUG S162- UGCCAGACACCUGACAUUAAGCAGA 162 UCUGCUUAAUGUCAGGUG 354 AS354-M1 UCUGGCAUG S163- CCUGACAUUAAGCAGAGCUUUUCCA 163 UGGAAAAGCUCUGCUUAA 355 AS355-M1 UGUCAGGUG S164- AAGCAGAGCUUUUCCAGAUAAAUTC 164 GAAUUUAUCUGGAAAAGC 356 AS356-M1 UCUGCUUAA S165- GCAGAGCUUUUCCAGAUAAAUUCCA 165 UGGAAUUUAUCUGGAAAA 357 AS357-M1 GCUCUGCUU S166- CCAGAUAAAUUCCAUGUGGAACUAA 166 UUAGUUCCACAUGGAAUU 358 AS358-M1 UAUCUGGAA S167- CAGAUAAAUUCCAUGUGGAACUAAC 167 GUUAGUUCCACAUGGAAU 359 AS359-M1 UUAUCUGGA S168- UCCUCAGUACCACCUUCUCCUUCAG 168 CUGAAGGAGAAGGUGGUA 360 AS360-M1 CUGAGGAAG S169- CCUCAGUACCACCUUCUCCUUCAGG 169 CCUGAAGGAGAAGGUGGU 361 AS361-M1 ACUGAGGAA S170- GAAAGGGAUCGGUUAAAUAUCAAGT 170 ACUUGAUAUUUAACCGAU 362 AS362-M1 CCCUUUCAG S171- AGGGAUCGGUUAAAUAUCAAGUCAC 171 GUGACUUGAUAUUUAACC 363 AS363-M1 GAUCCCUUU S172- AUCGGUUAAAUAUCAAGUCACCATT 172 AAUGGUGACUUGAUAUUU 364 AS364-M1 AACCGAUCC S173- GGUUAAAUAUCAAGUCACCAUUUTC 173 GAAAAUGGUGACUUGAUA 365 AS365-M1 UUUAACCGA S174- GUUAAAUAUCAAGUCACCAUUUUCA 174 UGAAAAUGGUGACUUGAU 366 AS366-M1 AUUUAACCG S175- AAAUAUCAAGUCACCAUUUUCACTG 175 CAGUGAAAAUGGUGACUU 367 AS367-M1 GAUAUUUAA S176- AAGAAAAAGCUGCAUGGUGAAUATA 176 UAUAUUCACCAUGCAGCU 368 AS368-M1 UUUUCUUCC S177- AGAAAAAGCUGCAUGGUGAAUAUAA 177 UUAUAUUCACCAUGCAGC 369 AS369-M1 UUUUUCUUC S178- AAAAAGCUGCAUGGUGAAUAUAAGA 178 UCUUAUAUUCACCAUGCA 370 AS370-M1 GCUUUUUCU S179- AAAAGCUGCAUGGUGAAUAUAAGAA 179 UUCUUAUAUUCACCAUGC 371 AS371-M1 AGCUUUUUC S180- AAGCUGCAUGGUGAAUAUAAGAACT 180 AGUUCUUAUAUUCACCAU 372 AS372-M1 GCAGCUUUU S181- GCUGCAUGGUGAAUAUAAGAACUGA 181 UCAGUUCUUAUAUUCACC 373 AS373-M1 AUGCAGCUU S182- CUGCAUGGUGAAUAUAAGAACUGAA 182 UUCAGUUCUUAUAUUCAC 374 AS374-M1 CAUGCAGCU S183- GCAUGGUGAAUAUAAGAACUGAATT 183 AAUUCAGUUCUUAUAUUC 375 AS375-M1 ACCAUGCAG S184- CAUGGUGAAUAUAAGAACUGAAUTC 184 GAAUUCAGUUCUUAUAUU 376 AS376-M1 CACCAUGCA S185- AUGGUGAAUAUAAGAACUGAAUUCT 185 AGAAUUCAGUUCUUAUAU 377 AS377-M1 UCACCAUGC S186- UGGUGAAUAUAAGAACUGAAUUCTA 186 UAGAAUUCAGUUCUUAUA 378 AS378-M1 UUCACCAUG S187- GGUGAAUAUAAGAACUGAAUUCUAC 187 GUAGAAUUCAGUUCUUAU 379 AS379-M1 AUUCACCAU S188- GUGAAUAUAAGAACUGAAUUCUACA 188 UGUAGAAUUCAGUUCUUA 380 AS380-M1 UAUUCACCA S189- AAUAUAAGAACUGAAUUCUACAUGT 189 ACAUGUAGAAUUCAGUUC 381 AS381-M1 UUAUAUUCA S190- AUAUAAGAACUGAAUUCUACAUGTG 190 CACAUGUAGAAUUCAGUU 382 AS382-M1 CUUAUAUUC S191- AUAAGAACUGAAUUCUACAUGUGCT 191 AGCACAUGUAGAAUUCAG 383 AS383-M1 UUCUUAUAU S192- AAGAACUGAAUUCUACAUGUGCUGC 192 GCAGCACAUGUAGAAUUCA 384 AS384-M1 GUUCUUAU S385- AGGUGCAUUUUGGAAGAUGGGCAGC 385 CCAUCUUCCAAAAUGCACC 417 AS417-M2 CGAAAGGCUGC UGG S385- AGGUGCAUUUUGGAAGAUGGGCAGC 385 CCAUCUUCCAAAAUGCACC 417 AS417-M3 CGAAAGGCUGC UGG S385- AGGUGCAUUUUGGAAGAUGGGCAGC 385 CCAUCUUCCAAAAUGCACC 417 AS417-M4 CGAAAGGCUGC UGG S385- AGGUGCAUUUUGGAAGAUGGGCAGC 385 CCAUCUUCCAAAAUGCACC 417 AS417-M5 CGAAAGGCUGC UGG S385- AGGUGCAUUUUGGAAGAUGGGCAGC 385 CCAUCUUCCAAAAUGCACC 417 AS417-M6 CGAAAGGCUGC UGG S385- AGGUGCAUUUUGGAAGAUGGGCAGC 385 CCAUCUUCCAAAAUGCACC 417 AS417-M7 CGAAAGGCUGC UGG S385- AGGUGCAUUUUGGAAGAUGGGCAGC 385 CCAUCUUCCAAAAUGCACC 417 AS417-M8 CGAAAGGCUGC UGG S385- AGGUGCAUUUUGGAAGAUGGGCAGC 385 CCAUCUUCCAAAAUGCACC 417 AS417-M9 CGAAAGGCUGC UGG S385- AGGUGCAUUUUGGAAGAUGGGCAGC 385 CCAUCUUCCAAAAUGCACC 417 AS417-M10 CGAAAGGCUGC UGG S385- AGGUGCAUUUUGGAAGAUGGGCAGC 385 CCAUCUUCCAAAAUGCACC 417 AS417-M11 CGAAAGGCUGC UGG S386- AGGUGCAUUUUGGAAGAUGGCAGCC 386 CAUCUUCCAAAAUGCACCU 418 AS418-M12 GAAAGGCUGC GG S386- AGGUGCAUUUUGGAAGAUGGCAGCC 386 CAUCUUCCAAAAUGCACCU 418 AS418-M13 GAAAGGCUGC GG S387- GGUGCAUUUUGGAAGAUGGCGCAGCC 387 GCCAUCUUCCAAAAUGCAC 419 AS419-M2 GAAAGGCUGC CUG S387- GGUGCAUUUUGGAAGAUGGCGCAGCC 387 GCCAUCUUCCAAAAUGCAC 419 AS419-M3 GAAAGGCUGC CUG S387- GGUGCAUUUUGGAAGAUGGCGCAGCC 387 GCCAUCUUCCAAAAUGCAC 419 AS419-M4 GAAAGGCUGC CUG S387- GGUGCAUUUUGGAAGAUGGCGCAGCC 387 GCCAUCUUCCAAAAUGCAC 419 AS419-M5 GAAAGGCUGC CUG S387- GGUGCAUUUUGGAAGAUGGCGCAGCC 387 GCCAUCUUCCAAAAUGCAC 419 AS419-M6 GAAAGGCUGC CUG S387- GGUGCAUUUUGGAAGAUGGCGCAGCC 387 GCCAUCUUCCAAAAUGCAC 419 AS419-M7 GAAAGGCUGC CUG S387- GGUGCAUUUUGGAAGAUGGCGCAGCC 387 GCCAUCUUCCAAAAUGCAC 419 AS419-M8 GAAAGGCUGC CUG S387- GGUGCAUUUUGGAAGAUGGCGCAGCC 387 GCCAUCUUCCAAAAUGCAC 419 AS419-M9 GAAAGGCUGC CUG S387- GGUGCAUUUUGGAAGAUGGCGCAGCC 387 GCCAUCUUCCAAAAUGCAC 419 AS419-M10 GAAAGGCUGC CUG S387- GGUGCAUUUUGGAAGAUGGCGCAGCC 387 GCCAUCUUCCAAAAUGCAC 419 AS419-M11 GAAAGGCUGC CUG S388- GGUGCAUUUUGGAAGAUGGGCAGCC 388 CCAUCUUCCAAAAUGCACC 420 AS420-M12 GAAAGGCUGC UG S388- GGUGCAUUUUGGAAGAUGGGCAGCC 388 CCAUCUUCCAAAAUGCACC 420 AS420-M13 GAAAGGCUGC UG S389- AAGGAAGUCCUUAUGUGGUAGCAGCC 389 UACCACAUAAGGACUUCCU 421 AS421-M2 GAAAGGCUGC UCU S389- AAGGAAGUCCUUAUGUGGUAGCAGCC 389 UACCACAUAAGGACUUCCU 421 AS421-M3 GAAAGGCUGC UCU S389- AAGGAAGUCCUUAUGUGGUAGCAGCC 389 UACCACAUAAGGACUUCCU 421 AS421-M4 GAAAGGCUGC UCU S389- AAGGAAGUCCUUAUGUGGUAGCAGCC 389 UACCACAUAAGGACUUCCU 421 AS421-M5 GAAAGGCUGC UCU S389- AAGGAAGUCCUUAUGUGGUAGCAGCC 389 UACCACAUAAGGACUUCCT 422 AS422-M6 GAAAGGCUGC UCU S389- AAGGAAGUCCUUAUGUGGUAGCAGCC 389 UACCACAUAAGGACUUCCU 421 AS421-M7 GAAAGGCUGC UCU S389- AAGGAAGUCCUUAUGUGGUAGCAGCC 389 UACCACAUAAGGACUUCCU 421 AS421-M8 GAAAGGCUGC UCU S389- AAGGAAGUCCUUAUGUGGUAGCAGCC 389 UACCACAUAAGGACUUCCU 421 AS421-M9 GAAAGGCUGC UCU S389- AAGGAAGUCCUUAUGUGGUAGCAGCC 389 UACCACAUAAGGACUUCCU 421 AS421-M10 GAAAGGCUGC UCU S389- AAGGAAGUCCUUAUGUGGUAGCAGCC 389 UACCACAUAAGGACUUCCU 421 AS421-M11 GAAAGGCUGC UCU S390- AAGGAAGUCCUUAUGUGGUGCAGCCG 390 ACCACAUAAGGACUUCCTU 423 AS423-M12 AAAGGCUGC CU S390- AAGGAAGUCCUUAUGUGGUGCAGCCG 390 ACCACAUAAGGACUUCCUU 424 AS424-M13 AAAGGCUGC CU S391- AUGAUAUGCUGAUAUUUGGAGCAGC 391 UCCAAAUAUCAGCAUAUCA 425 AS425-M2 CGAAAGGCUGC UUG S391- AUGAUAUGCUGAUAUUUGGAGCAGC 391 UCCAAAUAUCAGCAUAUCA 425 AS425-M3 CGAAAGGCUGC UUG S391- AUGAUAUGCUGAUAUUUGGAGCAGC 391 UCCAAAUAUCAGCAUAUCA 425 AS425-M4 CGAAAGGCUGC UUG S391- AUGAUAUGCUGAUAUUUGGAGCAGC 391 UCCAAAUAUCAGCAUAUCA 425 AS425-M5 CGAAAGGCUGC UUG S391- AUGAUAUGCUGAUAUUUGGAGCAGC 391 UCCAAAUAUCAGCAUATCA 426 AS426-M6 CGAAAGGCUGC UUG S391- AUGAUAUGCUGAUAUUUGGAGCAGC 391 UCCAAAUAUCAGCAUAUCA 425 AS425-M7 CGAAAGGCUGC UUG S391- AUGAUAUGCUGAUAUUUGGAGCAGC 391 UCCAAAUAUCAGCAUAUCA 425 AS425-M8 CGAAAGGCUGC UUG S391- AUGAUAUGCUGAUAUUUGGAGCAGC 391 UCCAAAUAUCAGCAUAUCA 425 AS425-M9 CGAAAGGCUGC UUG S391- AUGAUAUGCUGAUAUUUGGAGCAGC 391 UCCAAAUAUCAGCAUAUCA 425 AS425-M10 CGAAAGGCUGC UUG S391- AUGAUAUGCUGAUAUUUGGAGCAGC 391 UCCAAAUAUCAGCAUAUCA 425 AS425-M11 CGAAAGGCUGC UUG S392- AUGAUAUGCUGAUAUUUGGGCAGCC 392 CCAAAUAUCAGCAUATCAU 427 AS427-M12 GAAAGGCUGC UG S392- AUGAUAUGCUGAUAUUUGGGCAGCC 392 CCAAAUAUCAGCAUAUCAU 428 AS428-M13 GAAAGGCUGC UG S393- AGCAAAUAUUGAUUUCUACAGCAGCC 393 UGUAGAAAUCAAUAUUUG 429 AS429-M2 GAAAGGCUGC CUGC S393- AGCAAAUAUUGAUUUCUACAGCAGCC 393 UGUAGAAAUCAAUAUUUG 429 AS429-M3 GAAAGGCUGC CUGC S393- AGCAAAUAUUGAUUUCUACAGCAGCC 393 UGUAGAAAUCAAUAUUUG 429 AS429-M4 GAAAGGCUGC CUGC S393- AGCAAAUAUUGAUUUCUACAGCAGCC 393 UGUAGAAAUCAAUAUUUG 429 AS429-M5 GAAAGGCUGC CUGC S393- AGCAAAUAUUGAUUUCUACAGCAGCC 393 UGUAGAAAUCAATAUUTGC 430 AS430-M6 GAAAGGCUGC UGC S393- AGCAAAUAUUGAUUUCUACAGCAGCC 393 UGUAGAAAUCAAUAUUUG 429 AS429-M7 GAAAGGCUGC CUGC S393- AGCAAAUAUUGAUUUCUACAGCAGCC 393 UGUAGAAAUCAAUAUUUG 429 AS429-M8 GAAAGGCUGC CUGC S393- AGCAAAUAUUGAUUUCUACAGCAGCC 393 UGUAGAAAUCAAUAUUUG 429 AS429-M9 GAAAGGCUGC CUGC S393- AGCAAAUAUUGAUUUCUACAGCAGCC 393 UGUAGAAAUCAAUAUUUG 429 AS429-M10 GAAAGGCUGC CUGC S393- AGCAAAUAUUGAUUUCUACAGCAGCC 393 UGUAGAAAUCAAUAUUUG 429 AS429-M11 GAAAGGCUGC CUGC S394- AGCAAAUAUUGAUUUCUACGCAGCCG 394 GTAGAAAUCAATAUUTGCU 431 AS431-M12 AAAGGCUGC GC S394- AGCAAAUAUUGAUUUCUACGCAGCCG 394 GUAGAAAUCAAUAUUUGC 432 AS432-M13 AAAGGCUGC UGC S395- CCACGGUUUCUGAAAUAACAGCAGCC 395 UGUUAUUUCAGAAACCGU 433 AS433-M2 GAAAGGCUGC GGUG S395- CCACGGUUUCUGAAAUAACAGCAGCC 395 UGUUAUUUCAGAAACCGU 433 AS433-M3 GAAAGGCUGC GGUG S395- CCACGGUUUCUGAAAUAACAGCAGCC 395 UGUUAUUUCAGAAACCGU 433 AS433-M4 GAAAGGCUGC GGUG S395- CCACGGUUUCUGAAAUAACAGCAGCC 395 UGUUAUUUCAGAAACCGU 433 AS433-M5 GAAAGGCUGC GGUG S395- CCACGGUUUCUGAAAUAACAGCAGCC 395 UGUUAUUUCAGAAACCGU 433 AS433-M6 GAAAGGCUGC GGUG S395- CCACGGUUUCUGAAAUAACAGCAGCC 395 UGUUAUUUCAGAAACCGU 433 AS433-M7 GAAAGGCUGC GGUG S395- CCACGGUUUCUGAAAUAACAGCAGCC 395 UGUUAUUUCAGAAACCGU 433 AS433-M8 GAAAGGCUGC GGUG S395- CCACGGUUUCUGAAAUAACAGCAGCC 395 UGUUAUUUCAGAAACCGU 433 AS433-M9 GAAAGGCUGC GGUG S395- CCACGGUUUCUGAAAUAACAGCAGCC 395 UGUUAUUUCAGAAACCGU 433 AS433-M10 GAAAGGCUGC GGUG S395- CCACGGUUUCUGAAAUAACAGCAGCC 395 UGUUAUUUCAGAAACCGU 433 AS433-M11 GAAAGGCUGC GGUG S396- CCACGGUUUCUGAAAUAACGCAGCCG 396 GTUAUUUCAGAAACCGUG 434 AS434-M12 AAAGGCUGC GUG S396- CCACGGUUUCUGAAAUAACGCAGCCG 396 GUUAUUUCAGAAACCGUG 435 AS435-M13 AAAGGCUGC GUG S397- CUUGAAUGUUAAGAAAUUUUGCAGCC 397 AAAAUUUCUUAACAUUCAA 436 AS436-M2 GAAAGGCUGC GCC S397- CUUGAAUGUUAAGAAAUUUUGCAGCC 397 AAAAUUUCUUAACAUUCAA 436 AS436-M3 GAAAGGCUGC GCC S397- CUUGAAUGUUAAGAAAUUUUGCAGCC 397 AAAAUUUCUUAACAUUCAA 436 AS436-M4 GAAAGGCUGC GCC S397- CUUGAAUGUUAAGAAAUUUUGCAGCC 397 AAAAUUUCUUAACAUUCAA 436 AS436-M5 GAAAGGCUGC GCC S397- CUUGAAUGUUAAGAAAUUUUGCAGCC 397 AAAAUUUCUUAACAUUCAA 436 AS436-M6 GAAAGGCUGC GCC S397- CUUGAAUGUUAAGAAAUUUUGCAGCC 397 AAAAUUUCUUAACAUUCAA 436 AS436-M7 GAAAGGCUGC GCC S397- CUUGAAUGUUAAGAAAUUUUGCAGCC 397 AAAAUUUCUUAACAUUCAA 436 AS436-M8 GAAAGGCUGC GCC S397- CUUGAAUGUUAAGAAAUUUUGCAGCC 397 AAAAUUUCUUAACAUUCAA 436 AS436-M9 GAAAGGCUGC GCC S397- CUUGAAUGUUAAGAAAUUUUGCAGCC 397 AAAAUUUCUUAACAUUCAA 436 AS436-M10 GAAAGGCUGC GCC S397- CUUGAAUGUUAAGAAAUUUUGCAGCC 397 AAAAUUUCUUAACAUUCAA 436 AS436-M11 GAAAGGCUGC GCC S398- CUUGAAUGUUAAGAAAUUUGCAGCCG 398 AAAUUUCUUAACAUUCAA 437 AS437-M12 AAAGGCUGC GCC S398- CUUGAAUGUUAAGAAAUUUGCAGCCG 398 AAAUUUCUUAACAUUCAA 437 AS437-M13 AAAGGCUGC GCC S399- UGAAUGUUAAGAAAUUUUCAGCAGCC 399 UGAAAAUUUCUUAACAUU 438 AS438-M2 GAAAGGCUGC CAAG S399- UGAAUGUUAAGAAAUUUUCAGCAGCC 399 UGAAAAUUUCUUAACAUU 438 AS438-M3 GAAAGGCUGC CAAG S399- UGAAUGUUAAGAAAUUUUCAGCAGCC 399 UGAAAAUUUCUUAACAUU 438 AS438-M4 GAAAGGCUGC CAAG S399- UGAAUGUUAAGAAAUUUUCAGCAGCC 399 UGAAAAUUUCUUAACAUU 438 AS438-M5 GAAAGGCUGC CAAG S399- UGAAUGUUAAGAAAUUUUCAGCAGCC 399 UGAAAAUUUCUUAACATUC 439 AS439-M6 GAAAGGCUGC AAG S399- UGAAUGUUAAGAAAUUUUCAGCAGCC 399 UGAAAAUUUCUUAACAUU 438 AS438-M7 GAAAGGCUGC CAAG S399- UGAAUGUUAAGAAAUUUUCAGCAGCC 399 UGAAAAUUUCUUAACAUU 438 AS438-M8 GAAAGGCUGC CAAG S399- UGAAUGUUAAGAAAUUUUCAGCAGCC 399 UGAAAAUUUCUUAACAUU 438 AS438-M9 GAAAGGCUGC CAAG S399- UGAAUGUUAAGAAAUUUUCAGCAGCC 399 UGAAAAUUUCUUAACAUU 438 AS438-M10 GAAAGGCUGC CAAG S399- UGAAUGUUAAGAAAUUUUCAGCAGCC 399 UGAAAAUUUCUUAACAUU 438 AS438-M11 GAAAGGCUGC CAAG S400- UGAAUGUUAAGAAAUUUUCGCAGCCG 400 GAAAAUUUCUUAACATUCA 440 AS440-M12 AAAGGCUGC AG S400- UGAAUGUUAAGAAAUUUUCGCAGCCG 400 GAAAAUUUCUUAACAUUC 441 AS441-M13 AAAGGCUGC AAG S401- CGAGGUCAUUUCUAUGGUCAGCAGCC 401 UGACCAUAGAAAUGACCUC 442 AS442-M2 GAAAGGCUGC GAA S401- CGAGGUCAUUUCUAUGGUCAGCAGCC 401 UGACCAUAGAAAUGACCUC 442 AS442-M3 GAAAGGCUGC GAA S401- CGAGGUCAUUUCUAUGGUCAGCAGCC 401 UGACCAUAGAAAUGACCUC 442 AS442-M4 GAAAGGCUGC GAA S401- CGAGGUCAUUUCUAUGGUCAGCAGCC 401 UGACCAUAGAAAUGACCUC 442 AS442-M5 GAAAGGCUGC GAA S401- CGAGGUCAUUUCUAUGGUCAGCAGCC 401 UGACCAUAGAAATGACCUC 443 AS443-M6 GAAAGGCUGC GAA S401- CGAGGUCAUUUCUAUGGUCAGCAGCC 401 UGACCAUAGAAAUGACCUC 442 AS442-M7 GAAAGGCUGC GAA S401- CGAGGUCAUUUCUAUGGUCAGCAGCC 401 UGACCAUAGAAAUGACCUC 442 AS442-M8 GAAAGGCUGC GAA S401- CGAGGUCAUUUCUAUGGUCAGCAGCC 401 UGACCAUAGAAAUGACCUC 442 AS442-M9 GAAAGGCUGC GAA S401- CGAGGUCAUUUCUAUGGUCAGCAGCC 401 UGACCAUAGAAAUGACCUC 442 AS442-M10 GAAAGGCUGC GAA S401- CGAGGUCAUUUCUAUGGUCAGCAGCC 401 UGACCAUAGAAAUGACCUC 442 AS442-M11 GAAAGGCUGC GAA S402- CGAGGUCAUUUCUAUGGUCGCAGCCG 402 GACCAUAGAAATGACCUCG 444 AS444-M12 AAAGGCUGC AA S402- CGAGGUCAUUUCUAUGGUCGCAGCCG 402 GACCAUAGAAAUGACCUCG 445 AS445-M13 AAAGGCUGC AA S403- AUUCUGUGAAGGAAAAGUUUGCAGCC 403 AAACUUUUCCUUCACAGAA 446 AS446-M2 GAAAGGCUGC UGU S403- AUUCUGUGAAGGAAAAGUUUGCAGCC 403 AAACUUUUCCUUCACAGAA 446 AS446-M3 GAAAGGCUGC UGU S403- AUUCUGUGAAGGAAAAGUUUGCAGCC 403 AAACUUUUCCUUCACAGAA 446 AS446-M4 GAAAGGCUGC UGU S403- AUUCUGUGAAGGAAAAGUUUGCAGCC 403 AAACUUUUCCUUCACAGAA 446 AS446-M5 GAAAGGCUGC UGU S403- AUUCUGUGAAGGAAAAGUUUGCAGCC 403 AAACUUUUCCUUCACAGAA 446 AS446-M6 GAAAGGCUGC UGU S403- AUUCUGUGAAGGAAAAGUUUGCAGCC 403 AAACUUUUCCUUCACAGAA 446 AS446-M7 GAAAGGCUGC UGU S403- AUUCUGUGAAGGAAAAGUUUGCAGCC 403 AAACUUUUCCUUCACAGAA 446 AS446-M8 GAAAGGCUGC UGU S403- AUUCUGUGAAGGAAAAGUUUGCAGCC 403 AAACUUUUCCUUCACAGAA 446 AS446-M9 GAAAGGCUGC UGU S403- AUUCUGUGAAGGAAAAGUUUGCAGCC 403 AAACUUUUCCUUCACAGAA 446 AS446-M10 GAAAGGCUGC UGU S403- AUUCUGUGAAGGAAAAGUUUGCAGCC 403 AAACUUUUCCUUCACAGAA 446 AS446-M11 GAAAGGCUGC UGU S404- AUUCUGUGAAGGAAAAGUUGCAGCCG 404 AACUUUUCCUUCACAGAAU 447 AS447-M12 AAAGGCUGC GU S404- AUUCUGUGAAGGAAAAGUUGCAGCCG 404 AACUUUUCCUUCACAGAAU 447 AS447-M13 AAAGGCUGC GU S405- CUUGGAGUAUUUCCAUCAUAGCAGCC 405 UAUGAUGGAAAUACUCCA 448 AS448-M2 GAAAGGCUGC AGAU S405- CUUGGAGUAUUUCCAUCAUAGCAGCC 405 UAUGAUGGAAAUACUCCA 448 AS448-M3 GAAAGGCUGC AGAU S405- CUUGGAGUAUUUCCAUCAUAGCAGCC 405 UAUGAUGGAAAUACUCCA 448 AS448-M4 GAAAGGCUGC AGAU S405- CUUGGAGUAUUUCCAUCAUAGCAGCC 405 UAUGAUGGAAAUACUCCA 448 AS448-M5 GAAAGGCUGC AGAU S405- CUUGGAGUAUUUCCAUCAUAGCAGCC 405 UAUGAUGGAAAUACUCCA 448 AS448-M6 GAAAGGCUGC AGAU S405- CUUGGAGUAUUUCCAUCAUAGCAGCC 405 UAUGAUGGAAAUACUCCA 448 AS448-M7 GAAAGGCUGC AGAU S405- CUUGGAGUAUUUCCAUCAUAGCAGCC 405 UAUGAUGGAAAUACUCCA 448 AS448-M8 GAAAGGCUGC AGAU S405- CUUGGAGUAUUUCCAUCAUAGCAGCC 405 UAUGAUGGAAAUACUCCA 448 AS448-M9 GAAAGGCUGC AGAU S405- CUUGGAGUAUUUCCAUCAUAGCAGCC 405 UAUGAUGGAAAUACUCCA 448 AS448-M10 GAAAGGCUGC AGAU S405- CUUGGAGUAUUUCCAUCAUAGCAGCC 405 UAUGAUGGAAAUACUCCA 448 AS448-M11 GAAAGGCUGC AGAU S406- CUUGGAGUAUUUCCAUCAUGCAGCCG 406 ATGAUGGAAAUACUCCAAG 449 AS449-M12 AAAGGCUGC AU S406- CUUGGAGUAUUUCCAUCAUGCAGCCG 406 AUGAUGGAAAUACUCCAA 450 AS450-M13 AAAGGCUGC GAU S407- UGGAGUAUUUCCAUCAUACUGCAGCC 407 AGUAUGAUGGAAAUACUC 451 AS451-M2 GAAAGGCUGC CAAG S407- UGGAGUAUUUCCAUCAUACUGCAGCC 407 AGUAUGAUGGAAAUACUC 451 AS451-M3 GAAAGGCUGC CAAG S407- UGGAGUAUUUCCAUCAUACUGCAGCC 407 AGUAUGAUGGAAAUACUC 451 AS451-M4 GAAAGGCUGC CAAG S407- UGGAGUAUUUCCAUCAUACUGCAGCC 407 AGUAUGAUGGAAAUACUC 451 AS451-M5 GAAAGGCUGC CAAG S407- UGGAGUAUUUCCAUCAUACUGCAGCC 407 AGUAUGAUGGAAAUACTCC 452 AS452-M6 GAAAGGCUGC AAG S407- UGGAGUAUUUCCAUCAUACUGCAGCC 407 AGUAUGAUGGAAAUACUC 451 AS451-M7 GAAAGGCUGC CAAG S407- UGGAGUAUUUCCAUCAUACUGCAGCC 407 AGUAUGAUGGAAAUACUC 451 AS451-M8 GAAAGGCUGC CAAG S407- UGGAGUAUUUCCAUCAUACUGCAGCC 407 AGUAUGAUGGAAAUACUC 451 AS451-M9 GAAAGGCUGC CAAG S407- UGGAGUAUUUCCAUCAUACUGCAGCC 407 AGUAUGAUGGAAAUACUC 451 AS451-M10 GAAAGGCUGC CAAG S407- UGGAGUAUUUCCAUCAUACUGCAGCC 407 AGUAUGAUGGAAAUACUC 451 AS451-M11 GAAAGGCUGC CAAG S408- UGGAGUAUUUCCAUCAUACGCAGCCG 408 GTAUGAUGGAAAUACTCCA 453 AS453-M12 AAAGGCUGC AG S408- UGGAGUAUUUCCAUCAUACGCAGCCG 408 GUAUGAUGGAAAUACUCC 454 AS454-M13 AAAGGCUGC AAG S409- GAGUAUUUCCAUCAUACUAUGCAGCC 409 AUAGUAUGAUGGAAAUAC 455 AS455-M2 GAAAGGCUGC UCCA S409- GAGUAUUUCCAUCAUACUAUGCAGCC 409 AUAGUAUGAUGGAAAUAC 455 AS455-M3 GAAAGGCUGC UCCA S409- GAGUAUUUCCAUCAUACUAUGCAGCC 409 AUAGUAUGAUGGAAAUAC 455 AS455-M4 GAAAGGCUGC UCCA S409- GAGUAUUUCCAUCAUACUAUGCAGCC 409 AUAGUAUGAUGGAAAUAC 455 AS455-M5 GAAAGGCUGC UCCA S409- GAGUAUUUCCAUCAUACUAUGCAGCC 409 ATAGUAUGAUGGAAAUACT 456 AS456-M6 GAAAGGCUGC CCA S409- GAGUAUUUCCAUCAUACUAUGCAGCC 409 AUAGUAUGAUGGAAAUAC 455 AS455-M7 GAAAGGCUGC UCCA S409- GAGUAUUUCCAUCAUACUAUGCAGCC 409 AUAGUAUGAUGGAAAUAC 455 AS455-M8 GAAAGGCUGC UCCA S409- GAGUAUUUCCAUCAUACUAUGCAGCC 409 AUAGUAUGAUGGAAAUAC 455 AS455-M9 GAAAGGCUGC UCCA S409- GAGUAUUUCCAUCAUACUAUGCAGCC 409 AUAGUAUGAUGGAAAUAC 455 AS455-M10 GAAAGGCUGC UCCA S409- GAGUAUUUCCAUCAUACUAUGCAGCC 409 AUAGUAUGAUGGAAAUAC 455 AS455-M11 GAAAGGCUGC UCCA S410- GAGUAUUUCCAUCAUACUAGCAGCCG 410 UAGUAUGAUGGAAAUACT 457 AS457-M12 AAAGGCUGC CCA S410- GAGUAUUUCCAUCAUACUAGCAGCCG 410 UAGUAUGAUGGAAAUACU 458 AS458-M13 AAAGGCUGC CCA S411- CCAGCAUGCCAGACACCUGAGCAGCC 411 UCAGGUGUCUGGCAUGCU 459 AS459-M2 GAAAGGCUGC GGUA S411- CCAGCAUGCCAGACACCUGAGCAGCC 411 UCAGGUGUCUGGCAUGCU 459 AS459-M3 GAAAGGCUGC GGUA S411- CCAGCAUGCCAGACACCUGAGCAGCC 411 UCAGGUGUCUGGCAUGCU 459 AS459-M4 GAAAGGCUGC GGUA S411- CCAGCAUGCCAGACACCUGAGCAGCC 411 UCAGGUGUCUGGCAUGCU 459 AS459-M5 GAAAGGCUGC GGUA S411- CCAGCAUGCCAGACACCUGAGCAGCC 411 UCAGGUGUCUGGCAUGCU 459 AS459-M6 GAAAGGCUGC GGUA S411- CCAGCAUGCCAGACACCUGAGCAGCC 411 UCAGGUGUCUGGCAUGCU 459 AS459-M7 GAAAGGCUGC GGUA S411- CCAGCAUGCCAGACACCUGAGCAGCC 411 UCAGGUGUCUGGCAUGCU 459 AS459-M8 GAAAGGCUGC GGUA S411- CCAGCAUGCCAGACACCUGAGCAGCC 411 UCAGGUGUCUGGCAUGCU 459 AS459-M9 GAAAGGCUGC GGUA S411- CCAGCAUGCCAGACACCUGAGCAGCC 411 UCAGGUGUCUGGCAUGCU 459 AS459-M10 GAAAGGCUGC GGUA S411- CCAGCAUGCCAGACACCUGAGCAGCC 411 UCAGGUGUCUGGCAUGCU 459 AS459-M11 GAAAGGCUGC GGUA S412- CCAGCAUGCCAGACACCUGGCAGCCG 412 CAGGUGUCUGGCAUGCUG 460 AS460-M12 AAAGGCUGC GUA S412- CCAGCAUGCCAGACACCUGGCAGCCG 412 CAGGUGUCUGGCAUGCUG 460 AS460-M13A AAGGCUGC GUA S413- AGAAAAAGCUGCAUGGUGAAGCAGCC 413 UUCACCAUGCAGCUUUUU 461 AS461-M2 GAAAGGCUGC CUUC S413- AGAAAAAGCUGCAUGGUGAAGCAGCC 413 UUCACCAUGCAGCUUUUU 461 AS461-M3 GAAAGGCUGC CUUC S413- AGAAAAAGCUGCAUGGUGAAGCAGCC 413 UUCACCAUGCAGCUUUUU 461 AS461-M4 GAAAGGCUGC CUUC S413- AGAAAAAGCUGCAUGGUGAAGCAGCC 413 UUCACCAUGCAGCUUUUU 461 AS461-M5 GAAAGGCUGC CUUC S413- AGAAAAAGCUGCAUGGUGAAGCAGCC 413 UTCACCAUGCAGCUUUTUC 462 AS462-M6 GAAAGGCUGC UUC S413- AGAAAAAGCUGCAUGGUGAAGCAGCC 413 UUCACCAUGCAGCUUUUU 461 AS461-M7 GAAAGGCUGC CUUC S413- AGAAAAAGCUGCAUGGUGAAGCAGCC 413 UUCACCAUGCAGCUUUUU 461 AS461-M8 GAAAGGCUGC CUUC S413- AGAAAAAGCUGCAUGGUGAAGCAGCC 413 UUCACCAUGCAGCUUUUU 461 AS461-M9 GAAAGGCUGC CUUC S413- AGAAAAAGCUGCAUGGUGAAGCAGCC 413 UUCACCAUGCAGCUUUUU 461 AS461-M10 GAAAGGCUGC CUUC S413- AGAAAAAGCUGCAUGGUGAAGCAGCC 413 UUCACCAUGCAGCUUUUU 461 AS461-M11 GAAAGGCUGC CUUC S414- AGAAAAAGCUGCAUGGUGAGCAGCCG 414 UCACCAUGCAGCUUUTUCU 463 AS463-M12 AAAGGCUGC UC S414- AGAAAAAGCUGCAUGGUGAGCAGCCG 414 UCACCAUGCAGCUUUUUC 464 AS464-M13 AAAGGCUGC UUC S415- UGGUGAAUAUAAGAACUGAAGCAGCC 415 UUCAGUUCUUAUAUUCAC 465 AS465-M2 GAAAGGCUGC CAUG S415- UGGUGAAUAUAAGAACUGAAGCAGCC 415 UUCAGUUCUUAUAUUCAC 465 AS465-M3 GAAAGGCUGC CAUG S415- UGGUGAAUAUAAGAACUGAAGCAGCC 415 UUCAGUUCUUAUAUUCAC 465 AS465-M4 GAAAGGCUGC CAUG S415- UGGUGAAUAUAAGAACUGAAGCAGCC 415 UUCAGUUCUUAUAUUCAC 465 AS465-M5 GAAAGGCUGC CAUG S415- UGGUGAAUAUAAGAACUGAAGCAGCC 415 UTCAGUUCUUAUAUUCACC 598 AS598-M6 GAAAGGCUGC AUG S415- UGGUGAAUAUAAGAACUGAAGCAGCC 415 UUCAGUUCUUAUAUUCAC 465 AS465-M7 GAAAGGCUGC CAUG S415- UGGUGAAUAUAAGAACUGAAGCAGCC 415 UUCAGUUCUUAUAUUCAC 465 AS465-M8 GAAAGGCUGC CAUG S415- UGGUGAAUAUAAGAACUGAAGCAGCC 415 UUCAGUUCUUAUAUUCAC 465 AS465-M9 GAAAGGCUGC CAUG S415- UGGUGAAUAUAAGAACUGAAGCAGCC 415 UUCAGUUCUUAUAUUCAC 465 AS465-M10 GAAAGGCUGC CAUG S415- UGGUGAAUAUAAGAACUGAAGCAGCC 415 UUCAGUUCUUAUAUUCAC 465 AS465-M11 GAAAGGCUGC CAUG S416- UGGUGAAUAUAAGAACUGAGCAGCCG 416 UCAGUUCUUAUAUUCACC 466 AS466-M12 AAAGGCUGC AUG S416- UGGUGAAUAUAAGAACUGAGCAGCCG 416 UCAGUUCUUAUAUUCACC 466 AS466-M13 AAAGGCUGC AUG S467- GGAGGCAUCUAUACUGUGAUUCAGA 467 UCUGAAUCACAGUAUAGA 518 AS518-M1 UGCCUCCAA S468- GGCAUCUAUACUGUGAUUCAGACAA 468 UUGUCUGAAUCACAGUAU 519 AS519-M1 AGAUGCCUC S469- GCAUCUAUACUGUGAUUCAGACAAA 469 UUUGUCUGAAUCACAGUA 520 AS520-M1 UAGAUGCCU S470- GUCCAUAUUUUGAGCAUAAUAUGAA 470 UUCAUAUUAUGCUCAAAA 521 AS521-M1 UAUGGACCU S471- AUAUUUUGAGCAUAAUAUGAAGACT 471 AGUCUUCAUAUUAUGCUC 522 AS522-M1 AAAAUAUGG S52-AS244- GCAGCAAAUAUUGAUUUCUACAACC  52 GGUUGUAGAAAUCAAUAU 244 M1 UUGCUGCAC S55-AS247- GCAAAUAUUGAUUUCUACAACCATC  55 GAUGGUUGUAGAAAUCAA 247 M1 UAUUUGCUG S75-AS267- GGUUUCUGAAAUAACAGCAAUAGAA  75 UUCUAUUGCUGUUAUUUC 267 M1 AGAAACCGU S77-AS269- CUGAAAUAACAGCAAUAGAAGCUGA  77 UCAGCUUCUAUUGCUGUU 269 M1 AUUUCAGAA S472- ACGGCUUGAAUGUUAAGAAAUUUTC 472 GAAAAUUUCUUAACAUUC 523 AS523-M1 AAGCCGUUU S473- CGGCUUGAAUGUUAAGAAAUUUUCA 473 UGAAAAUUUCUUAACAUU 524 AS524-M1 CAAGCCGUU S80-AS272- GGCUUGAAUGUUAAGAAAUUUUCAG  80 CUGAAAAUUUCUUAACAU 272 M1 UCAAGCCGU S81-AS273- GCUUGAAUGUUAAGAAAUUUUCAGC  81 GCUGAAAAUUUCUUAACA 273 M1 UUCAAGCCG S82-AS274- CUUGAAUGUUAAGAAAUUUUCAGCA  82 UGCUGAAAAUUUCUUAAC 274 M1 AUUCAAGCC S83-AS275- UUGAAUGUUAAGAAAUUUUCAGCAG  83 CUGCUGAAAAUUUCUUAA 275 M1 CAUUCAAGC S84-AS276- UGAAUGUUAAGAAAUUUUCAGCAGT  84 ACUGCUGAAAAUUUCUUA 276 M1 ACAUUCAAG S85-AS277- GAAUGUUAAGAAAUUUUCAGCAGTG  85 CACUGCUGAAAAUUUCUU 277 M1 AACAUUCAA S474- AAUGUUAAGAAAUUUUCAGCAGUGC 474 GCACUGCUGAAAAUUUCU 525 AS525-M1 UAACAUUCA S86-AS278- AUGUUAAGAAAUUUUCAGCAGUGCA  86 UGCACUGCUGAAAAUUUC 278 M1 UUAACAUUC S475- UGUUAAGAAAUUUUCAGCAGUGCAT 475 AUGCACUGCUGAAAAUUU 526 AS526-M1 CUUAACAUU S476- GUUAAGAAAUUUUCAGCAGUGCATG 476 CAUGCACUGCUGAAAAUU 527 AS527-M1 UCUUAACAU S143- GGAGUAUUUCCAUCAUACUAUGAAC 143 GUUCAUAGUAUGAUGGAA 335 AS335-M1 AUACUCCAA S181- GCUGCAUGGUGAAUAUAAGAACUGA 181 UCAGUUCUUAUAUUCACC 373 AS373-M1 AUGCAGCUU S182- CUGCAUGGUGAAUAUAAGAACUGAA 182 UUCAGUUCUUAUAUUCAC 374 AS374-M1 CAUGCAGCU S477- UGCAUGGUGAAUAUAAGAACUGAAT 477 AUUCAGUUCUUAUAUUCA 528 AS528-M1 CCAUGCAGC S183- GCAUGGUGAAUAUAAGAACUGAATT 183 AAUUCAGUUCUUAUAUUC 375 AS375-M1 ACCAUGCAG S184- CAUGGUGAAUAUAAGAACUGAAUTC 184 GAAUUCAGUUCUUAUAUU 376 AS376-M1 CACCAUGCA S185- AUGGUGAAUAUAAGAACUGAAUUCT 185 AGAAUUCAGUUCUUAUAU 377 AS377-M1 UCACCAUGC S186- UGGUGAAUAUAAGAACUGAAUUCTA 186 UAGAAUUCAGUUCUUAUA 378 AS378-M1 UUCACCAUG S187- GGUGAAUAUAAGAACUGAAUUCUAC 187 GUAGAAUUCAGUUCUUAU 379 AS379-M1 AUUCACCAU S188- GUGAAUAUAAGAACUGAAUUCUACA 188 UGUAGAAUUCAGUUCUUA 380 AS380-M1 UAUUCACCA S478- UGAAUAUAAGAACUGAAUUCUACAT 478 AUGUAGAAUUCAGUUCUU 529 AS529-M1 AUAUUCACC S479- GAAUAUAAGAACUGAAUUCUACATG 479 CAUGUAGAAUUCAGUUCU 530 AS530-M1 UAUAUUCAC S189- AAUAUAAGAACUGAAUUCUACAUGT 189 ACAUGUAGAAUUCAGUUC 381 AS381-M1 UUAUAUUCA S480- CAAAGUAAGACUAAUUAUUUAAAAT 480 AUUUUAAAUAAUUAGUCU 531 AS531-M1 UACUUUGCU S481- AAAGUAAGACUAAUUAUUUAAAATA 481 UAUUUUAAAUAAUUAGUC 532 AS532-M1 UUACUUUGC S482- AGAAAUUGAGUGAAUGACAAUUUTG 482 CAAAAUUGUCAUUCACUCA 533 AS533-M1 AUUUCUUC S483- AAAUUGAGUGAAUGACAAUUUUGTA 483 UACAAAAUUGUCAUUCACU 534 AS534-M1 CAAUUUCU S484- AUUGAGUGAAUGACAAUUUUGUAAT 484 AUUACAAAAUUGUCAUUC 535 AS535-M1 ACUCAAUUU S485- AAUGACAAUUUUGUAAUUUAGGATA 485 UAUCCUAAAUUACAAAAUU 536 AS536-M1 GUCAUUCA S486- AAGUGUUUUUAAAAUGGUGAAUUTA 486 UAAAUUCACCAUUUUAAAA 537 AS537-M1 ACACUUUU S487- AGUGUUUUUAAAAUGGUGAAUUUAA 487 UUAAAUUCACCAUUUUAA 538 AS538-M1 AAACACUUU S488- CUUACUCUGUUUAUUUUUAAAUGAT 488 AUCAUUUAAAAAUAAACAG 539 AS539-M1 AGUAAGAG S489- CUCUGUUUAUUUUUAAAUGAUCATC 489 GAUGAUCAUUUAAAAAUA 540 AS540-M1 AACAGAGUA S490- UCUGUUUAUUUUUAAAUGAUCAUCA 490 UGAUGAUCAUUUAAAAAU 541 AS541-M1 AAACAGAGU S491- GUUUAUUUUUAAAUGAUCAUCAUAA 491 UUAUGAUGAUCAUUUAAA 542 AS542-M1 AAUAAACAG S492- AUCAUCAUAAUCCUUUGCUUACUAT 492 AUAGUAAGCAAAGGAUUA 543 AS543-M1 UGAUGAUCA S493- GUGCACUACCUACAUUUUUUAAATA 493 UAUUUAAAAAAUGUAGGU 544 AS544-M1 AGUGCACAU S494- GCUAGGUUUUUACUGAUUAUUUUCA 494 UGAAAAUAAUCAGUAAAAA 545 AS545-M1 CCUAGCUA S495- CUAGGUUUUUACUGAUUAUUUUCAT 495 AUGAAAAUAAUCAGUAAAA 546 AS546-M1 ACCUAGCU S496- AGGUUUUUACUGAUUAUUUUCAUTT 496 AAAUGAAAAUAAUCAGUAA 547 AS547-M1 AAACCUAG S497- CUGAUUAUUUUCAUUUUUCACAUGC 497 GCAUGUGAAAAAUGAAAA 548 AS548-M1 UAAUCAGUA S498- AUGGACAUUUAUGUCACUUUUGAAA 498 UUUCAAAAGUGACAUAAA 549 AS549-M1 UGUCCAUUA S499- GACAUUUAUGUCACUUUUGAAAUCT 499 AGAUUUCAAAAGUGACAU 550 AS550-M1 AAAUGUCCA S500- ACAUUUAUGUCACUUUUGAAAUCTA 500 UAGAUUUCAAAAGUGACA 551 AS551-M1 UAAAUGUCC S501- UAGAAUUGAUGUUGUAAUUAAUGCA 501 UGCAUUAAUUACAACAUCA 552 AS552-M1 AUUCUAGA S502- AGAAUUGAUGUUGUAAUUAAUGCAA 502 UUGCAUUAAUUACAACAUC 553 AS553-M1 AAUUCUAG S503- GAAUUGAUGUUGUAAUUAAUGCAAG 503 CUUGCAUUAAUUACAACAU 554 AS554-M1 CAAUUCUA S504- ACCAUCUUACUGUAACAUUUUUCTA 504 UAGAAAAAUGUUACAGUA 555 AS555-M1 AGAUGGUGG S505- CAUCUUACUGUAACAUUUUUCUATT 505 AAUAGAAAAAUGUUACAG 556 AS556-M1 UAAGAUGGU S506- UCUUACUGUAACAUUUUUCUAUUGT 506 ACAAUAGAAAAAUGUUACA 557 AS557-M1 GUAAGAUG S507- CUUACUGUAACAUUUUUCUAUUGTT 507 AACAAUAGAAAAAUGUUAC 558 AS558-M1 AGUAAGAU 5508- UUACUGUAACAUUUUUCUAUUGUTT 508 AAACAAUAGAAAAAUGUUA 559 AS559-M1 CAGUAAGA S509- ACUGUAACAUUUUUCUAUUGUUUAA 509 UUAAACAAUAGAAAAAUG 560 AS560-M1 UUACAGUAA S510- CUGUAACAUUUUUCUAUUGUUUAAA 510 UUUAAACAAUAGAAAAAU 561 AS561-M1 GUUACAGUA S511- UGUAACAUUUUUCUAUUGUUUAAAT 511 AUUUAAACAAUAGAAAAA 562 AS562-M1 UGUUACAGU S512- GUAACAUUUUUCUAUUGUUUAAATA 512 UAUUUAAACAAUAGAAAA 563 AS563-M1 AUGUUACAG S513- UAACAUUUUUCUAUUGUUUAAAUAG 513 CUAUUUAAACAAUAGAAAA 564 AS564-M1 AUGUUACA S514- AACAUUUUUCUAUUGUUUAAAUAGA 514 UCUAUUUAAACAAUAGAA 565 AS565-M1 AAAUGUUAC S515- ACAUUUUUCUAUUGUUUAAAUAGAA 515 UUCUAUUUAAACAAUAGA 566 AS566-M1 AAAAUGUUA S516- CAUUUUUCUAUUGUUUAAAUAGAAA 516 UUUCUAUUUAAACAAUAG 567 AS567-M1 AAAAAUGUU S517- GUCAAUCUUCAUAGAUGAUAACUTG 517 CAAGUUAUCAUCUAUGAA 568 AS568-M1 GAUUGACCA S569- GGCAUCUAUACUGUGAUUCAGCAGCC 569 UGAAUCACAGUAUAGAUG 575 AS575-M14 GAAAGGCUGC CCGG S569- GGCAUCUAUACUGUGAUUCAGCAGCC 569 UGAAUCACAGUAUAGAUG 575 AS575-M15 GAAAGGCUGC CCGG S570- GCAUCUAUACUGUGAUUCAGGCAGCC 570 CUGAAUCACAGUAUAGAU 576 AS576-M14 GAAAGGCUGC GCGG S570- GCAUCUAUACUGUGAUUCAGGCAGCC 570 CUGAAUCACAGUAUAGAU 576 AS576-M15 GAAAGGCUGC GCGG S571- GCAGCAAAUAUUGAUUUCUAGCAGCC 571 UAGAAAUCAAUAUUUGCU 577 AS577-M14 GAAAGGCUGC GCGG S571- GCAGCAAAUAUUGAUUUCUAGCAGCC 571 UAGAAAUCAAUAUUUGCU 577 AS577-M15 GAAAGGCUGC GCGG S572- CUGAAAUAACAGCAAUAGAAGCAGCC 572 UUCUAUUGCUGUUAUUUC 578 AS578-M15 GAAAGGCUGC AGGG S573- ACGGCUUGAAUGUUAAGAAAGCAGCC 573 UUUCUUAACAUUCAAGCC 579 AS579-M15 GAAAGGCUGC GUGG S574- AGUGUUUUUAAAAUGGUGAAGCAGC 574 UUCACCAUUUUAAAAACAC 580 AS580-M14 CGAAAGGCUGC UGG S574- AGUGUUUUUAAAAUGGUGAAGCAGC 574 UUCACCAUUUUAAAAACAC 580 AS580-M15 CGAAAGGCUGC UGG S385- AGGUGCAUUUUGGAAGAUGGGCAGC 385 CCAUCUUCCAAAAUGCACC 417 AS417-M16 CGAAAGGCUGC UGG S389- AAGGAAGUCCUUAUGUGGUAGCAGCC 389 UACCACAUAAGGACUUCCU 421 AS421-M17 GAAAGGCUGC UCU S391- AUGAUAUGCUGAUAUUUGGAGCAGC 391 UCCAAAUAUCAGCAUAUCA 425 AS425-M16 CGAAAGGCUGC UUG S393- AGCAAAUAUUGAUUUCUACAGCAGCC 393 UGUAGAAAUCAAUAUUUG 429 AS429-M16 GAAAGGCUGC CUGC S397- CUUGAAUGUUAAGAAAUUUUGCAGCC 397 AAAAUUUCUUAACAUUCAA 436 AS436-M16 GAAAGGCUGC GCC S397- CUUGAAUGUUAAGAAAUUUAGCAGCC 397 UAAAUUUCUUAACAUUCA 586 AS586-M18 GAAAGGCUGC AGGG S397- CUUGAAUGUUAAGAAAUUUUGCAGCC 397 AAAUUUCUUAACAUUCAA 587 AS587-M19 GAAAGGCUGC GGG S395- CCACGGUUUCUGAAAUAACAGCAGCC 395 UGUUAUUUCAGAAACCGU 433 AS433-M17 GAAAGGCUGC GGUG S399- UGAAUGUUAAGAAAUUUUCAGCAGCC 399 UGAAAAUUUCUUAACAUU 438 AS438-M16 GAAAGGCUGC CAAG S401- CGAGGUCAUUUCUAUGGUCAGCAGCC 401 UGACCAUAGAAAUGACCUC 442 AS442-M17 GAAAGGCUGC GAA S405- CUUGGAGUAUUUCCAUCAUAGCAGCC 405 UAUGAUGGAAAUACUCCA 448 AS448-M17 GAAAGGCUGC AGAU S413- AGAAAAAGCUGCAUGGUGAAGCAGCC 413 UUCACCAUGCAGCUUUUU 461 AS461-M17 GAAAGGCUGC CUUC S413- AGAAAAAGCUGCAUGGUGAAGCAGCC 413 UUCACCAUGCAGCUUUUU 588 AS588-M20 GAAAGGCUGC CUGG S413- AGAAAAAGCUGCAUGGUGAAGCAGCC 413 UCACCAUGCAGCUUUUUC 589 AS589-M21 GAAAGGCUGC UGG S413- AGAAAAAGCUGCAUGGUGAAGCAGCC 413 UUCACCAUGCAGCUUUUU 461 AS461-M22 GAAAGGCUGC CUUC S415- UGGUGAAUAUAAGAACUGAAGCAGCC 415 UUCAGUUCUUAUAUUCAC 465 AS465-M16 GAAAGGCUGC CAUG S393- AGCAAAUAUUGAUUUCUACAGCAGCC 393 UGUAGAAAUCAAUAUUUG 589 AS589-M18 GAAAGGCUGC CUGG S397- CUUGAAUGUUAAGAAAUUUAGCAGCC 397 UAAAUUUCUUAACAUUCA 586 AS586-M23 GAAAGGCUGC AGGG S399- UGAAUGUUAAGAAAUUUUCAGCAGCC 399 UGAAAAUUUCUUAACAUU 590 AS590-M18 GAAAGGCUGC CAGG S413- AGAAAAAGCUGCAUGGUGAAGCAGCC 413 UUCACCAUGCAGCUUUUU 588 AS588-M20 GAAAGGCUGC CUGG S415- UGGUGAAUAUAAGAACUGAAGCAGCC 415 UUCAGUUCUUAUAUUCAC 591 AS591-M18 GAAAGGCUGC CAGG S415- UGGUGAAUAUAAGAACUGAAGCAGCC 415 UUCAGUUCUUAUAUUCAC 591 AS591-M23 GAAAGGCUGC CAGG S397- CUUGAAUGUUAAGAAAUUUUGCAGCC 397 AAAAUUUCUUAACAUUCAA 592 AS592-M24 GAAAGGCUGC GGG S397- CUUGAAUGUUAAGAAAUUUUGCAGCC 397 AAAAUUUCUUAACAUUCAA 592 AS592-M25 GAAAGGCUGC GGG S397- CUUGAAUGUUAAGAAAUUUUGCAGCC 397 AAAAUUUCUUAACAUUCAA 592 AS592-M26 GAAAGGCUGC GGG S397- CUUGAAUGUUAAGAAAUUUUGCAGCC 397 AAAAUUUCUUAACAUUCAA 592 AS592-M27 GAAAGGCUGC GGG S399- UGAAUGUUAAGAAAUUUUCAGCAGCC 399 UGAAAAUUUCUUAACAUU 590 AS590-M24 GAAAGGCUGC CAGG S399- UGAAUGUUAAGAAAUUUUCAGCAGCC 399 UGAAAAUUUCUUAACAUU 590 AS590-M25 GAAAGGCUGC CAGG S399- UGAAUGUUAAGAAAUUUUCAGCAGCC 399 UGAAAAUUUCUUAACAUU 590 AS590-M26 GAAAGGCUGC CAGG S399- UGAAUGUUAAGAAAUUUUCAGCAGCC 399 UGAAAAUUUCUUAACAUU 590 AS590-M27 GAAAGGCUGC CAGG S415- UGGUGAAUAUAAGAACUGAAGCAGCC 415 UUCAGUUCUUAUAUUCAC 591 AS591-M28 GAAAGGCUGC CAGG S415- UGGUGAAUAUAAGAACUGAAGCAGCC 415 UUCAGUUCUUAUAUUCAC 591 AS591-M29 GAAAGGCUGC CAGG S415- UGGUGAAUAUAAGAACUGAAGCAGCC 415 UUCAGUUCUUAUAUUCAC 591 AS591-M30 GAAAGGCUGC CAGG S415- UGGUGAAUAUAAGAACUGAAGCAGCC 415 UUCAGUUCUUAUAUUCAC 591 AS591-M31 GAAAGGCUGC CAGG S397- CUUGAAUGUUAAGAAAUUUUGCAGCC 397 AAAAUUUCUUAACAUUCAA 592 AS592-M32 GAAAGGCUGC GGG S399- UGAAUGUUAAGAAAUUUUCAGCAGCC 399 UGAAAAUUUCUUAACAUU 590 AS590-M32 GAAAGGCUGC CAGG S415- UGGUGAAUAUAAGAACUGAAGCAGCC 415 UUCAGUUCUUAUAUUCAC 591 AS591-M33 GAAAGGCUGC CAGG S399- UGAAUGUUAAGAAAUUUUCAGCAGCC 399 UGAAAAUUUCUUAACAUU 590 AS590-M34 GAAAGGCUGC CAGG S399- UGAAUGUUAAGAAAUUUUCAGCAGCC 399 UGAAAAUUUCUUAACAUU 590 AS590-M35 GAAAGGCUGC CAGG S399- UGAAUGUUAAGAAAUUUUCAGCAGCC 399 UGAAAAUUUCUUAACAUU 590 AS590-M28 GAAAGGCUGC CAGG S399- UGAAUGUUAAGAAAUUUUCAGCAGCC 399 UGAAAAUUUCUUAACAUU 590 AS590-M36 GAAAGGCUGC CAGG S581- GAAUGUUAAGAAAUUUUCAGCAGCCG 581 UGAAAAUUUCUUAACAUU 593 AS593-M37 AAAGGCUGC CGG S399- UGAAUGUUAAGAAAUUUUCAGCAGCC 399 UGAAAAUUUCUUAACAUU 590 AS590-M38 GAAAGGCUGC CAGG S399- UGAAUGUUAAGAAAUUUUCAGCAGCC 399 UGAAAAUUUCUUAACAUU 590 AS590-M39 GAAAGGCUGC CAGG S399- UGAAUGUUAAGAAAUUUUCAGCAGCC 399 UGAAAAUUUCUUAACAUU 590 AS590-M40 GAAAGGCUGC CAGG S399- UGAAUGUUAAGAAAUUUUCAGCAGCC 399 UGAAAAUUUCUUAACAUU 590 AS590-M41 GAAAGGCUGC CAGG S399- UGAAUGUUAAGAAAUUUUCAGCAGCC 399 UGAAAAUUUCUUAACAUU 590 AS590-M42 GAAAGGCUGC CAGG S399- UGAAUGUUAAGAAAUUUUCAGCAGCC 399 UGAAAAUUUCUUAACAUU 590 AS590-M43 GAAAGGCUGC CAGG S399- UGAAUGUUAAGAAAUUUUCAGCAGCC 399 UGAAAAUUUCUUAACAUU 590 AS590-M24 GAAAGGCUGC CAGG S399- UGAAUGUUAAGAAAUUUUCAGCAGCC 399 UGAAAAUUUCUUAACAUU 590 AS590-M44 GAAAGGCUGC CAGG S399- UGAAUGUUAAGAAAUUUUCAGCAGCC 399 UGAAAAUUUCUUAACAUU 590 AS590-M28 GAAAGGCUGC CAGG S399- UGAAUGUUAAGAAAUUUUCAGCAGCC 399 UGAAAAUUUCUUAACAUU 590 AS590-M45 GAAAGGCUGC CAGG S399- UGAAUGUUAAGAAAUUUUCAGCAGCC 399 UGAAAAUUUCUUAACAUU 590 AS590-M40 GAAAGGCUGC CAGG S399- UGAAUGUUAAGAAAUUUUCAGCAGCC 399 UGAAAAUUUCUUAACAUU 590 AS590-M46 GAAAGGCUGC CAGG S399- UGAAUGUUAAGAAAUUUUCAGCAGCC 399 UGAAAAUUUCUUAACAUU 590 AS590-M38 GAAAGGCUGC CAGG S399- UGAAUGUUAAGAAAUUUUCAGCAGCC 399 UGAAAAUUUCUUAACAUU 590 AS590-M46 GAAAGGCUGC CAGG S571- GCAGCAAAUAUUGAUUUCUAGCAGCC 571 UAGAAAUCAAUAUUUGCU 577 AS577-M47 GAAAGGCUGC GCGG S573- ACGGCUUGAAUGUUAAGAAAGCAGCC 573 UUUCUUAACAUUCAAGCC 579 AS579-M47 GAAAGGCUGC GUGG S582- UGCAUGAGUUUCAAAAUCUAGCAGCC 582 UAGAUUUUGAAACUCAUG 594 AS594-M47 GAAAGGCUGC CAGG S583- UCGAGAUGAUCUAACAAUUAGCAGCC 583 UAAUUGUUAGAUCAUCUC 595 AS595-M47 GAAAGGCUGC GAGG S584- AGACUAAUUAUUUAAAAUAAGCAGCC 584 UUAUUUUAAAUAAUUAGU 596 AS596-M47 GAAAGGCUGC CUGG S574- AGUGUUUUUAAAAUGGUGAAGCAGC 574 UUCACCAUUUUAAAAACAC 580 AS580-M47 CGAAAGGCUGC UGG S585- AGAAUUGAUGUUGUAAUUAAGCAGCC 585 UUAAUUACAACAUCAAUUC 597 AS597-M47 GAAAGGCUGC UGG S399- UGAAUGUUAAGAAAUUUUCAGCAGCC 399 UGAAAAUUUCUUAACAUU 590 AS590-M48 GAAAGGCUGC CAGG S399- UGAAUGUUAAGAAAUUUUCAGCAGCC 399 UGAAAAUUUCUUAACAUU 590 AS590-M49 GAAAGGCUGC CAGG S399- UGAAUGUUAAGAAAUUUUCAGCAGCC 399 UGAAAAUUUCUUAACAUU 590 AS590-M50 GAAAGGCUGC CAGG S399- UGAAUGUUAAGAAAUUUUCAGCAGCC 399 UGAAAAUUUCUUAACAUU 590 AS590-M51 GAAAGGCUGC CAGG S399- UGAAUGUUAAGAAAUUUUCAGCAGCC 399 UGAAAAUUUCUUAACAUU 590 AS590-M52 GAAAGGCUGC CAGG S399- UGAAUGUUAAGAAAUUUUCAGCAGCC 399 UGAAAAUUUCUUAACAUU 590 AS590-M53 GAAAGGCUGC CAGG S399- UGAAUGUUAAGAAAUUUUCAGCAGCC 399 UGAAAAUUUCUUAACAUU 590 AS590-M54 GAAAGGCUGC CAGG S399- UGAAUGUUAAGAAAUUUUCAGCAGCC 399 UGAAAAUUUCUUAACAUU 590 AS590-M55 GAAAGGCUGC CAGG S573- ACGGCUUGAAUGUUAAGAAAGCAGCC 573 UUUCUUAACAUUCAAGCC 579 AS579-M56 GAAAGGCUGC GUGG S573- ACGGCUUGAAUGUUAAGAAAGCAGCC 573 UUUCUUAACAUUCAAGCC 579 AS579-M57 GAAAGGCUGC GUGG S573- ACGGCUUGAAUGUUAAGAAAGCAGCC 573 UUUCUUAACAUUCAAGCC 579 AS579-M50 GAAAGGCUGC GUGG S573- ACGGCUUGAAUGUUAAGAAAGCAGCC 573 UUUCUUAACAUUCAAGCC 579 AS579-M51 GAAAGGCUGC GUGG S573- ACGGCUUGAAUGUUAAGAAAGCAGCC 573 UUUCUUAACAUUCAAGCC 579 AS579-M52 GAAAGGCUGC GUGG S573- ACGGCUUGAAUGUUAAGAAAGCAGCC 573 UUUCUUAACAUUCAAGCC 579 AS579-M53 GAAAGGCUGC GUGG S573- ACGGCUUGAAUGUUAAGAAAGCAGCC 573 UUUCUUAACAUUCAAGCC 579 AS579-M54 GAAAGGCUGC GUGG S573- ACGGCUUGAAUGUUAAGAAAGCAGCC 573 UUUCUUAACAUUCAAGCC 579 AS579-M55 GAAAGGCUGC GUGG S583- UCGAGAUGAUCUAACAAUUAGCAGCC 583 UAAUUGUUAGAUCAUCUC 595 AS595-M56 GAAAGGCUGC GAGG S583- UCGAGAUGAUCUAACAAUUAGCAGCC 583 UAAUUGUUAGAUCAUCUC 595 AS595-M58 GAAAGGCUGC GAGG S583- UCGAGAUGAUCUAACAAUUAGCAGCC 583 UAAUUGUUAGAUCAUCUC 595 AS595-M50 GAAAGGCUGC GAGG S583- UCGAGAUGAUCUAACAAUUAGCAGCC 583 UAAUUGUUAGAUCAUCUC 595 AS595-M51 GAAAGGCUGC GAGG S583- UCGAGAUGAUCUAACAAUUAGCAGCC 583 UAAUUGUUAGAUCAUCUC 595 AS595-M52 GAAAGGCUGC GAGG S583- UCGAGAUGAUCUAACAAUUAGCAGCC 583 UAAUUGUUAGAUCAUCUC 595 AS595-M53 GAAAGGCUGC GAGG S583- UCGAGAUGAUCUAACAAUUAGCAGCC 583 UAAUUGUUAGAUCAUCUC 595 AS595-M54 GAAAGGCUGC GAGG S583- UCGAGAUGAUCUAACAAUUAGCAGCC 583 UAAUUGUUAGAUCAUCUC 595 AS595-M55 GAAAGGCUGC GAGG S583- UCGAGAUGAUCUAACAAUUAGCAGCC 583 UAAUUGUUAGAUCAUCUC 595 AS595-M59 GAAAGGCUGC GAGG S583- UCGAGAUGAUCUAACAAUUAGCAGCC 583 UAAUUGUUAGAUCAUCUC 595 AS595-M60 GAAAGGCUGC GAGG S583- UCGAGAUGAUCUAACAAUUAGCAGCC 583 UAAUUGUUAGAUCAUCUC 595 AS595-M61 GAAAGGCUGC GAGG S583- UCGAGAUGAUCUAACAAUUAGCAGCC 583 UAAUUGUUAGAUCAUCUC 595 AS595-M62 GAAAGGCUGC GAGG S583- UCGAGAUGAUCUAACAAUUAGCAGCC 583 UAAUUGUUAGAUCAUCUC 595 AS595-M63 GAAAGGCUGC GAGG S583- UCGAGAUGAUCUAACAAUUAGCAGCC 583 UAAUUGUUAGAUCAUCUC 595 AS595-M64 GAAAGGCUGC GAGG S583- UCGAGAUGAUCUAACAAUUAGCAGCC 583 UAAUUGUUAGAUCAUCUC 595 AS595-M65 GAAAGGCUGC GAGG S583- UCGAGAUGAUCUAACAAUUAGCAGCC 583 UAAUUGUUAGAUCAUCUC 595 AS595-M66 GAAAGGCUGC GAGG S574- AGUGUUUUUAAAAUGGUGAAGCAGC 574 UUCACCAUUUUAAAAACAC 580 AS580-M56 CGAAAGGCUGC UGG S574- AGUGUUUUUAAAAUGGUGAAGCAGC 574 UUCACCAUUUUAAAAACAC 580 AS580-M58 CGAAAGGCUGC UGG S574- AGUGUUUUUAAAAUGGUGAAGCAGC 574 UUCACCAUUUUAAAAACAC 580 AS580-M50 CGAAAGGCUGC UGG S574- AGUGUUUUUAAAAUGGUGAAGCAGC 574 UUCACCAUUUUAAAAACAC 580 AS580-M51 CGAAAGGCUGC UGG S574- AGUGUUUUUAAAAUGGUGAAGCAGC 574 UUCACCAUUUUAAAAACAC 580 AS580-M52 CGAAAGGCUGC UGG S574- AGUGUUUUUAAAAUGGUGAAGCAGC 574 UUCACCAUUUUAAAAACAC 580 AS580-M53 CGAAAGGCUGC UGG S574- AGUGUUUUUAAAAUGGUGAAGCAGC 574 UUCACCAUUUUAAAAACAC 580 AS580-M54 CGAAAGGCUGC UGG S574- AGUGUUUUUAAAAUGGUGAAGCAGC 574 UUCACCAUUUUAAAAACAC 580 AS580-M55 CGAAAGGCUGC UGG S612- UGGGAGGUAUGAGUUUUCAAGCAGC 612 UUGAAAACUCAUACCUCCC 620 AS620-M5 CGAAAGGCUGC AGG S613- AGGAAAAGUUUGGAAAAAAAGCAGCC 613 UUUUUUUCCAAACUUUUC 621 AS621-M5 GAAAGGCUGC CUGG S614- UGGUCAAUCUUCAUAGAUGAGCAGCC 614 UCAUCUAUGAAGAUUGAC 622 AS622-M5 GAAAGGCUGC CAGG S615- GGUUUCUGAAAUAACAGCAAGCAGCC 615 UUGCUGUUAUUUCAGAAA 623 AS623-M5 GAAAGGCUGC CCGG S572- CUGAAAUAACAGCAAUAGAAGCAGCC 572 UUCUAUUGCUGUUAUUUC 578 AS578-M5 GAAAGGCUGC AGGG S616- GUGAAUAUAAGAACUGAAUUGCAGCC 616 AAUUCAGUUCUUAUAUUC 624 AS624-M5 GAAAGGCUGC ACGG S617- AUUGAGUGAAUGACAAUUUUGCAGCC 617 AAAAUUGUCAUUCACUCAA 625 AS625-M5 GAAAGGCUGC UGG S618- AAUGACAAUUUUGUAAUUUAGCAGCC 618 UAAAUUACAAAAUUGUCA 626 AS626-M5 GAAAGGCUGC UUGG S585- AGAAUUGAUGUUGUAAUUAAGCAGCC 585 UUAAUUACAACAUCAAUUC 597 AS597-M5 GAAAGGCUGC UGG S619- GAAUUGAUGUUGUAAUUAAUGCAGC 619 AUUAAUUACAACAUCAAUU 627 AS627-M5 CGAAAGGCUGC CGG S573- ACGGCUUGAAUGUUAAGAAAGCAGCC 573 UUUCUUAACAUUCAAGCC 579 AS579-M5 GAAAGGCUGC GUGG S582- UGCAUGAGUUUCAAAAUCUAGCAGCC 582 UAGAUUUUGAAACUCAUG 594 AS594-M5 GAAAGGCUGC CAGG S583- UCGAGAUGAUCUAACAAUUAGCAGCC 583 UAAUUGUUAGAUCAUCUC 595 AS595-M5 GAAAGGCUGC GAGG S584- AGACUAAUUAUUUAAAAUAAGCAGCC 584 UUAUUUUAAAUAAUUAGU 596 AS596-M5 GAAAGGCUGC CUGG

The disclosure illustratively described herein suitably can be practiced in the absence of any element or elements, limitation or limitations that are not specifically disclosed herein. Thus, for example, in each instance herein any of the terms “comprising”, “consisting essentially of”, and “consisting of” may be replaced with either of the other two terms. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments, optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the description and the appended claims.

In addition, where features or aspects of the invention are described in terms of Markush groups or other grouping of alternatives, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group or other group.

It should be appreciated that, in some embodiments, sequences presented in the sequence listing may be referred to in describing the structure of an oligonucleotide or other nucleic acid. In such embodiments, the actual oligonucleotide or other nucleic acid may have one or more alternative nucleotides (e.g., an RNA counterpart of a DNA nucleotide or a DNA counterpart of an RNA nucleotide) and/or one or more modified nucleotides and/or one or more modified internucleotide linkages and/or one or more other modification compared with the specified sequence while retaining essentially same or similar complementary properties as the specified sequence.

The use of the terms “a” and “an” and “the” and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms “comprising,” “having,” “including,” and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to,”) unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

Embodiments of this invention are described herein. Variations of those embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description.

The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims. 

What is claimed is:
 1. An oligonucleotide for reducing expression of GYS2, the oligonucleotide comprising an antisense strand of 15 to 30 nucleotides in length and a sense strand of 36 to 40 nucleotides in length, wherein the sense strand forms a duplex region with the antisense strand, and wherein: the antisense strand has a region of complementarity to a target sequence of GYS2 as set forth in any one of SEQ ID NOs: 599-608, wherein the region of complementarity is at least 15 contiguous nucleotides in length; and the sense strand comprises a sequence as set forth in any one of SEQ ID NOs: 385-416, 569-574, 581-585, and 612-619.
 2. The oligonucleotide of claim 1, wherein the antisense strand comprises a sequence as set forth in any one of SEQ ID NOs: 417-466, 575-580, 586-598, and 620-627.
 3. The oligonucleotide of claim 1, wherein the sense strand comprises at its 3′-end a stem-loop set forth as: S₁-L-S₂, wherein S₁ is complementary to S₂, and wherein L forms a loop between S₁ and S₂ of 3 to 5 nucleotides in length.
 4. The oligonucleotide of claim 1, wherein the oligonucleotide comprises a 3′-overhang sequence of one or more nucleotides in length, wherein the 3′-overhang sequence is present on the antisense strand, the sense strand, or the antisense strand and sense strand.
 5. The oligonucleotide of claim 1, wherein the oligonucleotide comprises at least one modified nucleotide and wherein the modified nucleotide comprises a 2′-modification selected from: 2′-aminoethyl, 2′-fluoro, 2′-O-methyl, 2′-O-methoxyethyl, and 2′-deoxy-2′-fluoro-β-d-arabinonucleic acid.
 6. The oligonucleotide of claim 1, wherein the oligonucleotide comprises at least one modified internucleotide linkage, wherein the at least one modified internucleotide linkage is a phosphorothioate linkage.
 7. The oligonucleotide of claim 1, wherein the duplex region formed by the sense strand and the antisense strand is at least 19 nucleotides in length.
 8. The oligonucleotide of claim 1, wherein the region of complementarity of the antisense strand to the target sequence of GYS2 is at least 19 contiguous nucleotides in length.
 9. The oligonucleotide of claim 1, wherein the antisense strand is 21 to 27 nucleotides in length.
 10. The oligonucleotide of claim 1, wherein at least one nucleotide of the oligonucleotide is conjugated to one or more targeting ligands, wherein each targeting ligand comprises a N-acetylgalactosamine (GalNAc) moiety.
 11. The oligonucleotide of claim 10, wherein the GalNac moiety is a monovalent GalNAc moiety, a bivalent GalNAc moiety, a trivalent GalNAc moiety, or a tetravalent GalNAc moiety.
 12. The oligonucleotide of claim 1, wherein the 4′-carbon of the sugar of the 5′-nucleotide of the antisense strand comprises a phosphate analog, and wherein the phosphate analog is oxymethylphosphonate, vinylphosphonate, or malonylphosphonate.
 13. A composition comprising the oligonucleotide of claim 1 and an excipient.
 14. An oligonucleotide for reducing expression of GYS2, the oligonucleotide comprising an antisense strand of 15 to 30 nucleotides in length and a sense strand of 15 to 40 nucleotides in length, wherein the sense strand forms a duplex region with the antisense strand, and wherein: the antisense strand has a region of complementarity to a target sequence of GYS2 as set forth in any one of SEQ ID NOs: 599-608, wherein the region of complementarity is at least 15 contiguous nucleotides in length; and the sense strand comprises at its 3′-end a stem-loop set forth as: S₁-L-S₂, wherein S₁ is complementary to S₂; L forms a loop between S₁ and S₂; and L is a tetraloop.
 15. The oligonucleotide of claim 14, wherein the antisense strand comprises a sequence as set forth in any one of SEQ ID NOs: 417-466, 575-580, 586-598, and 620-627.
 16. The oligonucleotide of claim 14, wherein the sense strand comprises a sequence as set forth in any one of SEQ ID NOs: 385-416, 569-574, 581-585, and 612-619.
 17. The oligonucleotide of claim 14, wherein the duplex region formed by the sense strand and the antisense strand is at least 19 nucleotides in length.
 18. The oligonucleotide of claim 14, wherein the region of complementarity of the antisense strand to the target sequence of GYS2 is at least 19 contiguous nucleotides in length.
 19. The oligonucleotide of claim 14, wherein the antisense strand is 21 to 27 nucleotides in length.
 20. The oligonucleotide of claim 14, wherein the oligonucleotide comprises a 3′-overhang sequence of one or more nucleotides in length, wherein the 3′-overhang sequence is present on the antisense strand, the sense strand, or the antisense strand and sense strand.
 21. The oligonucleotide of claim 14, wherein the oligonucleotide comprises at least one modified nucleotide and wherein the modified nucleotide comprises a 2′-modification selected from: 2′-aminoethyl, 2′-fluoro, 2′-O-methyl, 2′-O-methoxyethyl, and 2′-deoxy-2′-fluoro-β-d-arabinonucleic acid.
 22. The oligonucleotide of claim 14, wherein the oligonucleotide comprises at least one modified internucleotide linkage, wherein the at least one modified internucleotide linkage is a phosphorothioate linkage.
 23. The oligonucleotide of claim 14, wherein at least one nucleotide of the oligonucleotide is conjugated to one or more targeting ligands, wherein each targeting ligand comprises a N-acetylgalactosamine (GalNAc) moiety.
 24. The oligonucleotide of claim 23, wherein the GalNac moiety is a monovalent GalNAc moiety, a bivalent GalNAc moiety, a trivalent GalNAc moiety, or a tetravalent GalNAc moiety.
 25. The oligonucleotide of claim 14, wherein the 4′-carbon of the sugar of the 5′-nucleotide of the antisense strand comprises a phosphate analog, and wherein the phosphate analog is oxymethylphosphonate, vinylphosphonate, or malonylphosphonate.
 26. A composition comprising the oligonucleotide of claim 14 and an excipient.
 27. An oligonucleotide for reducing expression of GYS2, wherein: the antisense strand has a region of complementarity to a target sequence of GYS2 as set forth in any one of SEQ ID NOs: 599-608, wherein the region of complementarity is at least 15 contiguous nucleotides in length; the oligonucleotide comprises a 3′-overhang sequence of two nucleotides in length, wherein the 3′-overhang sequence is present on the antisense strand; the sense strand is 21 nucleotides in length and the antisense strand is 23 nucleotides in length, such that the sense strand and antisense strand form a duplex of 21 nucleotides in length; the oligonucleotide comprises at least one modified nucleotide and wherein the modified nucleotide comprises a 2′-modification selected from: 2′-aminoethyl, 2′-fluoro, 2′-O-methyl, 2′-O-methoxyethyl, and 2′-deoxy-2′-fluoro-β-d-arabinonucleic acid; and wherein the 4′-carbon of the sugar of the 5′-nucleotide of the antisense strand comprises a phosphate analog, and wherein the phosphate analog is oxymethylphosphonate, vinylphosphonate, or malonylphosphonate.
 28. The oligonucleotide of claim 27, wherein at least one nucleotide of the oligonucleotide is conjugated to one or more targeting ligands, wherein each targeting ligand comprises a N-acetylgalactosamine (GalNAc) moiety.
 29. The oligonucleotide of claim 28, wherein the GalNac moiety is a monovalent GalNAc moiety, a bivalent GalNAc moiety, a trivalent GalNAc moiety, or a tetravalent GalNAc moiety.
 30. The oligonucleotide of claim 27, wherein the duplex region formed by the sense strand and the antisense strand is at least 19 nucleotides in length.
 31. The oligonucleotide of claim 27, wherein the region of complementarity of the antisense strand to the target sequence of GYS2 is at least 19 contiguous nucleotides in length.
 32. The oligonucleotide of claim 27, wherein the oligonucleotide comprises at least one modified internucleotide linkage, wherein the at least one modified internucleotide linkage is a phosphorothioate linkage.
 33. The oligonucleotide of claim 27, wherein at least one nucleotide of the oligonucleotide is conjugated to one or more targeting ligands, wherein each targeting ligand comprises a N-acetylgalactosamine (GalNAc) moiety.
 34. The oligonucleotide of claim 33, wherein the GalNac moiety is a monovalent GalNAc moiety, a bivalent GalNAc moiety, a trivalent GalNAc moiety, or a tetravalent GalNAc moiety.
 35. A composition comprising the oligonucleotide of claim 27 and an excipient. 